CN101627128A - Bladder cancer biomarkers and uses thereof - Google Patents

Abstract

The invention relates to the identification and selection of novel biomarkers and the identification and selection of novel biomarker combinations which are differentially expressed in individuals with bladder cancer as compared with individuals without bladder cancer. Polynucleotides and proteins which specifically and/or selectively hybridize to the products of the biomarkers of the invention are also encompassed within the scope of the invention as are kits containing said polynucleotides and proteins for use in diagnosing bladder cancer. Further encompassed by the invention is the use of the polynucleotides and proteins which specifically and/or selectively hybridize to the product of the biomarkers of the invention to monitor disease regression in an individual and to monitor the efficacy of therapeutic regimens. The invention also provides for methods of using the products of the biomarkers of the invention in the identification of novel therapeutic targets for bladder cancer.

Description

Bladder cancer biomarkers and uses thereof

The application requires the U.S. Provisional Application 60/676,921 submitted on May 2nd, 2005 and the rights and interests of the U.S. Provisional Application 60/729,056 submitted on October 21st, 2005, and it is by with reference to incorporating this paper into.

The application comprises CD, duplicate (2 CDs: table-copy 1 and table-copy 2), and its full content is incorporated this paper into by reference.Every CD is identical and contains following file (corresponding to table 1-7 and 11-12):

1. technical field

The present invention includes and identify and select new bladder cancer biomarkers, it comprises early stage bladder cancer biomarkers, and identify and select new biomarker combination, compare with the individuality that does not have bladder cancer, the individual difference with bladder cancer or superficial bladder cancer is expressed described new biomarker combination.The product of measuring the combination of biomarker of the present invention and biomarker shows, has special advantage aspect the bladder cancer diagnosing individuality to have.The present invention also comprise specificity and/or optionally with the product hybridization/bonded polynucleotide and the protein of biomarker of the present invention.The present invention also provides the method for using biomarker product authenticating compound of the present invention, described compound combination and/or the activity of regulating biomarker gene of the present invention.Can be used for development research bladder cancer and bladder cancer analysis of Development method through this method compounds identified.In addition, can be used as the lead compound of developing preventative and therapeutic composition, be used for prevention, treatment, control and/or alleviation bladder cancer or its symptom through this method compounds identified.

2. background technology

Bladder cancer is the 4th cancer of normal diagnosis among the male sex, and is the 8th cancer of diagnosing out the most normal (Greenlee et al., Cancer Statistics, 2001) in the female patient.Proved that being exposed to cigarette, aromatic amine, coal combustion by product, chlorinated cpds and some aldehyde compound environment increases the risk that people suffer from bladder cancer.Bladder cancer self is lower to the influence of mortality ratio, yet this cancer has significantly increased mortality risk to the transfer of other position.Therefore, identify that the diagnosis of bladder cancer method will be useful.Particularly, allow the diagnostic method of early detection and intervention will have great benefit.

The bladder cancer screening method of current application comprises the application cystoscopy; Bladder irrigation and/or urinary cytology inspection.Each of these methods allows the doctor check that bladder and/or bladder cell are to detect tumour or other bladder cellular abnormality.Yet these methods are that cost is huge, and therefore for the people with the increase of bladder cancer risk not under a cloud, these methods are unpractiaca as screening method.Another habitual triage techniques is blood urine analysis (that is the blood in the inspection urine).Although the blood urine modal symptom that is bladder cancer, it also may indicate many other potential diagnosis (that is low specificity).For example, in the prospective analysis of Britain, have only whole blood urine patients of 19.2% to be determined and have bladder cancer by cytoscopy to the Hematuria Clinic patient.In addition, these methods are to those patients of early stage bladder cancer even have lower specificity and susceptibility.

Therefore, need method relatively cheap and enough sensitivities and specific screening bladder cancer in the art, to improve detection to those patients with bladder cancer risk.Also need to have the method for the early stage relatively bladder cancer of bigger ability screening, to allow to finding that those patients that suffer from bladder cancer implement to interfere in early days and more treatment selection.

3. summary of the invention

The combination of identifying and selecting new bladder cancer biomarkers and evaluation and selection shallow (in early days) bladder cancer biomarkers and identify these biomarkers is contained in the present invention, thereby simple and relatively cheap check is provided, and then the improvement to the bladder cancer screening is provided.Method disclosed herein and use biomarker that these methods identify and the product of the combination of biomarker proves, they have special advantage the screening patient aspect the patient who identifies the bladder cancer risk and increase.Should be understood that, in order to measure the product of biomarker of the present invention, the product of the biomarker of identifying with the present invention and derivative specificity thereof and/or selective cross/bonded polynucleotide and protein are also contained within the scope of the invention, contain described polynucleotide and protein and are used to diagnose the test kit of suffering from the bladder cancer individuality like this equally.In addition, the purposes that is used for monitoring the effect of development of individuality bladder cancer and monitor therapy scheme with the polynucleotide of the product specificity of biomarker of the present invention and/or selective cross and protein is also contained in the present invention.The present invention also provides the product of using biomarker of the present invention to be used to identify the authentication method of the new treatment target of bladder cancer.

3.1 definition

The definition of concrete term below is provided, and it is used for later written explanation.

As used herein, term " 3 ' end " is meant the end of mRNA, mostly be most 1/3 of last 1000 Nucleotide or described mRNA, wherein 3 ' terminal nucleotide and (might the exist) coding of poly A tract adjacency or the terminal nucleotide of non-translational region.That is to say that the 3 ' end of mRNA does not include the poly A tract that may exist." 3 ' district " of gene be meant and be positioned at gene 3 ' end place or its polynucleotide (two strands or strand), includes but not limited to the 3 ' non-translational region that might exist and 3 ' protein coding region of gene.3 ' district is not shorter than 8 Nucleotide length and is no more than 1000 Nucleotide long.3 ' distinguishes other possible length includes but not limited to 10,20,25,50,100,200,400 and 500 Nucleotide.

As used herein, term " 5 ' end " is meant the end of the mRNA that begins from first Nucleotide of described mRNA, mostly is 1/3 (total length of wherein said mRNA does not comprise poly-A tail) of preceding 1000 Nucleotide of described mRNA or described mRNA most." 5 ' district " of gene be meant and be positioned at gene 5 ' end place or its polynucleotide (two strands or strand), and include but not limited to the 5 ' non-translational region that might exist and 5 ' protein coding region of gene.Described 5 ' district is not shorter than 8 Nucleotide length and is no more than 1000 Nucleotide long.Described 5 ' distinguishes other possible length includes but not limited to 10,20,25,50,100,200,400 and 500 Nucleotide.

As used herein, term " amplification " is when being used for nucleotide sequence, be meant a kind of method, produce one or the specific nucleic acid sequence of multiple copied from template nucleic acid thus, preferably implement (Mullis and Faloona by the method for polymerase chain reaction, 1987, Method Enzymol., 155:335)." polymerase chain reaction " or " PCR " is meant the in vitro method of the specific nucleic acid template sequence that is used to increase.In some embodiments, the PCR reaction comprises a series of multiple temperature cycle and implements with the volume of 50-100 μ l usually.Reaction mixture comprises various dNTP (each of four kinds of deoxynucleotide dATP, dCTP, dGTP and dTTP), primer, damping fluid, archaeal dna polymerase and nucleic acid-templated.The PCR reaction can comprise provides one group of polynucleotide primer, wherein first primer contains the sequence that is complementary to zone in the described nucleic acid-templated chain and causes synthetic complementary dna chain, second primer contains the sequence that is complementary in described target nucleic acid sequence second chain zone and causes synthetic complementary dna chain, and use nucleic acid polymerase as the template dependency polymerizing agent described nucleic acid-templated sequence that increases, it is implemented under allowing the condition of following PCR circulation step: contained target nucleic acid sequence is annealed, (ii) extend described primer, wherein nucleic acid polymerase synthetic primer extension products." one group of polynucleotide primer " or " one group of PCR primer " can comprise two kinds, three kinds, four kinds or multiple primer.In one embodiment, use exo-Pfu archaeal dna polymerase amplification of nucleic acid template in the PCR reaction.Other amplification method includes but not limited to ligase chain reaction (LCR) (LCR), based on the specific amplification of polynucleotide (polynucleotide-specificbased amplication) (NSBA) or any known other method in this area.

As used herein, " N-terminal " of polypeptide district is meant by being positioned at mRNA 5 ' end or its polynucleotide sequence (two strands or strand) encoded polypeptides sequence.As used herein, " N-terminal " district is meant the N-terminal of the polypeptide that begins from first amino acid of described polypeptide, mostly is 1/3 of preceding 300 amino acid of described polypeptide or described polypeptide most." N-terminal " district of polypeptide is not shorter than 3 amino acid longs and is no more than 350 amino acid longs.Other of polypeptide " N-terminal " district may length include but not limited to 5,10,20,25,50,100 and 200 amino acid.

As used herein, in the protein agent (for example, protein, polypeptide, peptide and antibody) category in, term " analogue " is meant the protein agent that has or identical function similar to the second protein agent but might not comprise or same acid sequence similar to the second protein agent, perhaps has the protein agent of or same structure similar to the second protein agent.Protein agent with similar aminoacid sequence is meant satisfies following at least one the second protein agent: the protein agent that (a) has the aminoacid sequence of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% consistence with the aminoacid sequence of the second protein agent; (b) under rigorous condition with the coding second protein agent at least 5 continuous amino acid residues, at least 10 continuous amino acid residues, at least 15 continuous amino acid residues, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous amino acid residues, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, the protein agent that the nucleotide sequence of the nucleotide sequence hybridization of at least 125 continuous amino acid residues or at least 150 continuous amino acid residues is coded; (c) with the nucleotide sequence coded protein agent of nucleotide sequence with at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% consistence of the coding second protein agent.Be meant the protein agent that has similar secondary, three grades or quaternary structure to the described second protein agent to protein agent that the second protein agent has an analog structure.Can determine the structure of protein agent by method known to those skilled in the art, described method includes but not limited to peptide sequencing, X-radiocrystallography, nucleus magnetic resonance, circular dichroism and crystal current sub-microscope.

For determining the consistence percentage of two aminoacid sequences or two nucleotide sequences, for the best comparison purpose, described sequence is compared (for example, can introduce breach in first aminoacid sequence or nucleotide sequence is used for comparing with the best of second amino acid or nucleotide sequence).Subsequently amino-acid residue or Nucleotide at corresponding amino acid position or nucleotide position are compared.When identical amino-acid residue or Nucleotide were occupied on the corresponding position in by described second sequence position in described first sequence, so described molecule was consistent on this position.Consistence percentage between described two sequences is the total consistent positional number purpose function (that is lap position number/total number of positions * 100% of consistence %=unanimity) of described sequence.In one embodiment, the equal in length of described two sequences.

Can also finish consistence between two sequences percentile definite by the applied mathematics algorithm.Be used for that of mathematical algorithm of two sequence alignments is preferred, non-limiting example is following algorithm: Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.U.S.A.87:2264-2268, by Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.U.S.A.90:5873-5877 improves.This algorithm is integrated into Altschul et al., and 1990, in the NBLAST of J.Mol.Biol.215:403 and the XBLAST program.The BLAST nucleotide search can be implemented with NBLAST Nucleotide program, and described program parameter is set to, and for example scoring=100, word length=12 are to obtain and nucleic acid molecule homologous nucleotide sequence of the present invention.Can implement the BLAST protein search with the XBLAST program, described program parameter is set to, and for example scoring-50, word length=3 are to obtain and protein molecule homologous aminoacid sequence of the present invention.For obtaining to be used to compare the breach comparison of purpose, can utilize breach BLAST, as Altschul et al., 1997, described in the Nucleic Acids Res.25:3389-3402.As an alternative, can use PSI-BLAST and implement iterative search, the edge relation far away (Id.) between its detection molecules.When using BLAST, breach BLAST and PSI-Blast program, can use each program () default parameter (seeing, for example the NCBI website) for example, XBLAST and NALAST.Another of mathematical algorithm that is used for sequence alignment is preferred, non-limiting example is Myers and Miller, and 1988, the described algorithm of CABIOS 4:11-17.This algorithm is integrated in ALIGN program (2.0 version), and it is the part of GCG sequence alignment software package.When using the ALIGN program and be used to compare aminoacid sequence, can use PAM120 weighting residue table, notch length point penalty be 12 and the breach point penalty be 4.Can use and above-mentioned similar technology,, determine two consistence percentage between the sequence allowing or not allowing under the condition of breach.When calculating the consistence percentage, only consider the accurately situation of coupling usually.

As used herein, in the category of nonprotein analogue, term " analogue " is meant to have organic to first or inorganic molecule is similar or identical function and the second organic or inorganic molecule organic to described first or that inorganic molecule is structurally similar.

As used herein, term " biomarker " is meant gene, compare the gene of differential expression in individuality with the individuality (although individuality can have other disease) of the bladder cancer that does not have bladder cancer or certain phase, and can comprise the gene of comparing differential expression in having the individuality of superficial bladder cancer with the individuality that does not have bladder cancer with the bladder cancer or the bladder cancer in described stage.

Term used herein " biomarker Auele Specific Primer " is meant one group of primer, and it can produce a part of complementary double-stranded DNA with one or more RNA products of biomarker of the present invention.For example, described primer can comprise can selective cross and biomarker complementary RNA of the present invention, cDNA or EST to produce first primer of extension products, and second primer that can the described extension products of selective cross, it is used to produce and biomarker complementary double-stranded DNA of the present invention.

Term used herein " biomarker specific probe " is meant and the RNA selectivity of product of unique biomarker and the probe of specific hybrid.In one embodiment, the biomarker specific probe can be the probe with fluorophore and quencher, for example

Probe or Molecular

Probe.In another embodiment, the biomarker specific probe be attached to array and with the probe of one or more RNA products of unique biomarker cDNA, EST or the PCR product of described RNA product (perhaps corresponding to) selectivity and specific hybrid.The biomarker specific probe can comprise oligonucleotide probe and can comprise longer probe (for example, 100,150,200,250,300,350,400,450,500 Nucleotide etc.).

As used herein, term " bladder cancer " is meant a kind of disease, and wherein the cell of intravesical lining has been lost the ability of regulating and control its growth, causes forming the cell mass of tumour thus.As used herein, term " early stage bladder cancer " or " superficial bladder cancer " are meant Non-Invasive bladder cancer (for example, Ta or the Tia type of determining according to the AJCC guide) (Herr et al., 2001).

As used herein, term " blood nucleic acid samples " is meant the nucleic acid that comes from blood and can comprises and come from whole blood, centrifugal cracking blood, serum-free whole blood or the classification blood nucleic acid of (comprising peripheral blood leucocyte (PBL) or other blood ingredient as described herein).The whole blood meaning is meant unassorted blood and comprises drop of blood that wherein drop of blood comprises the volume of 5 μ l, 10 μ l, 15 μ l, 20 μ l, 25 μ l, 30 μ l.The meaning of centrifugal cracking blood or " cracking blood " is unassorted whole blood, and it is mixed with lysis buffer and as described hereinly carries out centrifugal (seeing embodiment 2).The serum-free blood meaning is meant unassorted whole blood, centrifugal removal serum wherein as described herein or blood plasma (seeing embodiment 2).Preferably, the blood nucleic acid samples is whole blood or centrifugal cracking blood and is total RNA, mRNA or corresponding to the nucleic acid of mRNA, for example isolating cDNA from described blood.Nucleic acid samples can also comprise the PCR product that comes from total RNA, mRNA or cDNA.

As used herein, " C-terminal " of polypeptide district is meant by being positioned at mRNA 3 ' end or its polynucleotide sequence (two strands or strand) encoded polypeptides sequence.As used herein, " C-terminal " district is meant the C-terminal of polypeptide, mostly is most from 1/3 of 300 amino acid of the described polypeptide of described polypeptide final amino acid meter or described polypeptide." 3 ' end " do not include the poly-A tail that may exist." C-terminal " district of polypeptide is not shorter than 3 amino acid longs and is no more than 350 amino acid longs.Other of polypeptide " C-terminal " district may length include but not limited to 5,10,20,25,50,100 and 200 amino acid.

As used herein, term " tissue sample " is meant the cell that comes from tissue, and described organizing is not blood, and can comprise bladder body, cerebral tissue, heart tissue, lung tissue, lymphoglandula etc." tissue nucleic acid samples " is total RNA, mRNA or corresponding to the nucleic acid of RNA, for example cDNA.The PCR product of organizing nucleic acid samples to comprise to come from total RNA, mRNA or cDNA.

As used herein, term " combination of biomarker of the present invention " is meant as the disclosed any or multiple biomarker of table 1, table 3, table 4, table 6, table 7 and table 10, table 11 or table 12.Biomarker combination of the present invention also comprises any combination as table 1 and the disclosed any two kinds or more of biomarkers of table 2, as long as at least a biomarker of described combination is from table 1.Biomarker combination of the present invention also comprises any combination as table 4 and disclosed any two or more biomarkers of table 5, as long as at least a biomarker of described combination is from table 4.Biomarker combination of the present invention also comprises any combination as table 11 and disclosed any two or more biomarkers of table 2, as long as at least a biomarker of described combination is from table 11.

As used herein, term " compound " and " reagent " are used interchangeably.

As used herein, " substantially by ... (the consisting essentially of) of composition " is meant the maximum number of the biomarker that can be used to diagnose or screen bladder cancer and/or diagnosis or screening superficial bladder cancer.In one embodiment, the biomarker that is used for diagnosing bladder cancer and/or superficial bladder cancer substantially by table 1 biomarker at least arbitrarily, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200,225,250 or all described biomarkers form.In another embodiment, the biomarker that is used for diagnosing bladder cancer substantially by table 7 biomarker any at least, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200 or all described biomarkers form.In another embodiment, the biomarker that is used for diagnosing bladder cancer substantially by table 10 biomarker any at least, maximum 1,2,3,4,5,6,7,8,9 or all described biomarkers form.In another embodiment, the biomarker that is used for diagnosing bladder cancer substantially at least by table 1 biomarker at least arbitrarily, maximum 1,2,3,4,5,6,7,8,9,10,11,13,15,20,25,30,35,40,45,50,100,125,150,175, any at least in 200 described biomarkers and the disclosed biomarker of PCT application No.PCT/US04/020836, maximum 1,2,3,4,5,6,7,8,9 or 10,15,20,30,40,50,100,200,300,400,500,1000,1500,2000,2500,3000,3500,4000 or all described biomarker combinations and form, the described biomarker of this PCT application provides in table 2.In another embodiment, the biomarker that is used for diagnosing shallow or commitment bladder cancer substantially by table 4 biomarker at least arbitrarily, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200,225,250,275,300,325,350,375,400,425,450,475,500 or all described biomarkers form.In another embodiment, the biomarker that is used for diagnosing superficial bladder cancer substantially by table 4 biomarker at least arbitrarily, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200,225,250,275,300,325,350,375,400,425,450,475, in 500 or all described biomarker and the disclosed biomarker of PCT application No.PCT/US04/020836 at least arbitrarily, maximum 1,2,3,4,5,6,7,8,9 or 10,15,20,30,40,50,100,150,200,250,300,350,400,450,500,1000,1500,2000,2500 or all described biomarker combinations and form, the described biomarker of this PCT application provides in table 5.In another embodiment, the biomarker that is used for diagnosing bladder cancer and/or superficial bladder cancer substantially by table 11 biomarker any at least, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200,225,250 or all described biomarkers form.In another embodiment, the biomarker that is used for diagnosing bladder cancer and/or superficial bladder cancer substantially by table 12 biomarker any at least, maximum 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,100,125,150,175,200,225,250 or all described biomarkers form.

As used herein, in category of the present invention, term " contrast " or " control sample " are meant that one or more organize nucleic acid samples and/or blood nucleic acid samples and/or one or more individuality, and described individuality is classified as to have prostate cancer, have the bladder cancer in one or more stages and comprise superficial bladder cancer; Do not have bladder cancer or do not have the bladder cancer (for example superficial bladder cancer) of certain phase; (for example, being used for detecting urine center stroma protein (is called wherein to use those technology well known by persons skilled in the art

) the level rising

Intravenously pyloegram (IVP) imaging, CT scan, MRI scanning, bone scanning or ultrasonic etc.).Term contrast or control sample can also refer to come from the compilation of the data of one or more individualities, and described individuality is diagnosed as normally (does not have bladder cancer), have bladder cancer or have the bladder cancer (for example superficial bladder cancer) of certain phase.Those skilled in the art are to be understood that term contrast is to be used in the category of test and will to depend on desirable comparison.As used herein, term " contrast " is meant in the category of the preventative or therapeutic agent of screening and is used to measure or the standard or the reference of method that other condition can compare with it.

As used herein, term " data " or " biomarkcr data " typically refer to the abundance of reflection biomarker product or the data of level, and described product comprises by one of the RNA of biomarker coding and protein or two kinds.

As used herein, term " derivative " in the protein agent (for example, protein, polypeptide, peptide and antibody) category in be meant the protein agent, it comprises the reformed aminoacid sequence by introducing one or more amino-acid residue replacements, disappearance and/or interpolation.Term used herein " derivative " also refers to adorned protein agent,, the molecule of any kind is covalently attached to described protein agent that is.For example; in unrestriced mode, can be by for example glycosylation, acetylize, Pegylation, phosphorylation, amidation, by known protection/blocking groups derivatize, proteolysis cutting, be connected in modified antibodies such as cell ligand or other protein.Can be by using technology well known in the art, including but not limited to that specificity chemical chop, acetylize, formylation, tunicamycin metabolism are synthetic etc. carries out chemically modified and produces the derivative of protein agent.In addition, protein agent derivative can comprise one or more atypical amino acid.Protein agent derivative have to from the similar or identical functions of its deutero-protein agent.

As used herein, term " derivative " is meant the second organic or inorganic molecule that forms based on the first organic or inorganic molecular structure in the category of nonprotein derivative.The organic molecule derivative include but not limited to by, for example, add or remove hydroxyl, methyl, ethyl, carboxyl or the amino molecule of being modified.Organic molecule can also be esterified, alkylation and/or phosphorylation.

" bladder cancer diagnosis " used herein or " diagnosing bladder cancer ", " screening bladder cancer ", " screening bladder cancer stage " are meant according to an aspect of the present invention determines that the people has bladder cancer and/or has the process of superficial bladder cancer possibility.Described term is meant diagnosis or screening bladder cancer or the conventional medical diagnostic techniqu in bladder cancer stage, and diagnosis and/or the screening method contained as the present invention.The conventional medical diagnostic techniqu of diagnosing bladder cancer comprises physical examination and medical history, medical evaluation, comprises urine examination and suitable laboratory inspection, and it can comprise usefulness

Detecting urine center stroma protein (is known as

) level rising, intravenously pyloegram (IVP) imaging, CT scan, MRI scanning, bone scanning or ultrasonic etc.Concrete an enforcement in the embodiment, " diagnosing prostate cancer " is meant between two options and determines: for example individual bladder cancer or the individual bladder cancer that does not have bladder cancer or specified phase with bladder cancer or specified phase.In a specific embodiments, term " diagnosis or screening bladder cancer " is meant between two options and determines, for example for " diagnosing bladder cancer ", determines that individuality has bladder cancer or the individual possibility that does not have bladder cancer.For " diagnosis superficial bladder cancer ", determine that individuality has superficial bladder cancer or do not have the possibility of bladder cancer.In another embodiment, " diagnosis " is meant determining between three options, and one of them option is to determine that individuality has bladder cancer or do not have bladder cancer (for example determining that the result is uncertain) with enough degree.In another embodiment, " diagnosis or screening bladder cancer " can comprise can not with enough degree determine individuality be can be characterized by have bladder cancer or a specified phase bladder cancer or the option that does not have bladder cancer or specified phase bladder cancer.One skilled in the art will appreciate that in this category " enough degree are determined " depends on medically to diagnostic sensitivity and specific requirement.More particularly, enough degree are determined to comprise greater than 50% susceptibility and/or specificity, greater than 60% susceptibility and/or specificity, greater than 70% susceptibility and/or specificity, greater than 80% susceptibility and/or specificity, greater than 90% susceptibility and/or specificity and 100% susceptibility and/or specificity.

As used herein, measure RNA that term " differential expression " is meant at one or more biomarkers of the present invention and/or the difference on the protein expression level by RNA or proteinic amount or level.Can be included in RNA (one or more splice variants that comprise mRNA and/or described mRNA) the correlated difference of expression level of the expression level of the mRNA of biomarker described in the sample and/or one or more described mRNA splice variants and one or more biomarkers of identical the present invention of amount by measure R NA or level determination in second sample with regard to RNA." differential expression " or " differential expression " can also comprise in sample of measurement or the sample colony by biomarker encoded protein matter of the present invention or one or more protein variant, and with the amount of protein expression or level relatively, described protein comprises one or more protein variant of biomarker of the present invention.Can be according to of the present invention and understood by one of ordinary skill in the artly determine differential expression.Term " differential expression " or " expression level variation " are meant the expression level measured and increase or the reduction compared at the expression level measured of biomarker specific product described in second sample of the biomarker specific product of measuring by measure R NA amount and protein mass in a sample.Described first sample and second sample need be from different patients, and can be the samples of gathering from same patient on different time points.Term " differential expression " or " changes of expression level " can also refer to the expression level measured and increase or the reduction compared at the expression level measured of biomarker described in other sample colonies of biomarker-specific in some sample colonies.As used herein, " differential expression " is when the expression simple sample, can be applied in the ratio that the expression level of biomarker-specific in the described sample compares with the average expression level of control population biomarker-specific and measure, wherein said ratio is not equal to 1.0.Can also the application of difference expression comprise with second colony of first colony of some samples and some samples or with simple sample and sample colony comparing, wherein use the ratio of expression level or use the p-value.When using the p-value, measurement to the significance,statistical of transcribed nucleic acid thing (comprising hnRNA and mRNA) differential expression is the differential expression of identifying between first and second colonies, when the p-value less than 0.3,0.2,0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001 etc. the time, think to have significance,statistical.When determining differential expression based on the ratio of gene product expression level, if compare with second sample, the ratio of its RNA or protein level is greater than or less than 1.0 in first sample, and RNA or protein gene product are by differential expression.For example, the RNA of gene or the ratio of protein be greater than 1, for example 1.2,1.5,1.7,2,3,4,10,20 or ratio less than 1, for example the 0.8,0.6,0.4,0.2,0.1, the 0.05th, the index of differential expression.In another embodiment of the present invention, if the expression mean level (ML) of first transcript is greater than or less than 1.0 with its ratio of expressing mean level (ML) in second colony in nucleic acid population, transcribed nucleic acid thing (comprising hnRNA and mRNA) is by differential expression.For example, ratio is greater than 1, for example 1.2,1.5,1.7,2,3,4,10,20 or ratio less than 1, for example 0.8,0.6,0.4,0.2,0.1,0.05 will be the index of differential expression.In another embodiment of the present invention, if the ratio of the mean value of its expression level and second colony is greater than or less than 1.0 and for example comprise in first sample, ratio is greater than 1, for example 1.2,1.5,1.7,2,3,4,10,20 or ratio less than 1, for example 0.8,0.6,0.4,0.2,0.1,0.05, transcribed nucleic acid thing (comprising hnRNA and mRNA) is by differential expression." otherness increases expression " is meant 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times or bigger of relative standard's (such as mean value of second colony's expression level)." otherness reduces expression " is meant that relative standard's (such as mean value of second colony's expression level) is less than 1.0 times, 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times or littler.

As used herein, term " efficacy of drugs " is meant the validity of medicine.Usually by the mensuration of accepting or just accepting pharmacological agent patient's clinical response is measured " efficacy of drugs ".If medicine has reached the clinical effectiveness of expection, for example, alleviated the symptom of osteoarthritis as described in this manual or prevented the development of osteoarthritis, just think that medicine has the effect of height.The absorbed dose of medicine can be used to predict reaction.General rule is when drug dose increases, and sees bigger effect in the patient, until reaching the greatest expected effect.If use more medicine after reaching maximum point, side effect will increase usually.

As used herein, term " significant quantity " is meant development and/or the seriousness that is enough to alleviate or alleviate bladder cancer or its one or more symptoms, the development that prevents bladder cancer or its one or more symptoms, recurrence or outbreak, prevents bladder cancer or its one or more severity of symptoms or strengthens or improve the compound amount of the preventative or therapeutic effect of another therapy.

As used herein, term " fragment " is meant peptide or polypeptide in the category of protein agent, it comprises polypeptide or proteinic at least 5 continuous amino acid residues, at least 10 continuous amino acid residues, at least 15 continuous amino acid residues, at least 20 continuous amino acid residues, at least 25 continuous amino acid residues, at least 40 continuous amino acid residues, at least 50 continuous amino acid residues, at least 60 continuous amino acid residues, at least 70 continuous amino acid residues, at least 80 continuous amino acid residues, at least 90 continuous amino acid residues, at least 100 continuous amino acid residues, at least 125 continuous amino acid residues, at least 150 continuous amino acid residues, at least 175 continuous amino acid residues, the aminoacid sequence of at least 200 continuous amino acid residues or at least 250 continuous amino acid residues.In a specific embodiments, the fragment of protein or polypeptide keeps at least a function of described protein or polypeptide.In another embodiment, the fragment of protein or polypeptide keeps at least a, two kinds, three kinds, four kinds or five kinds of functions of described protein or polypeptide.Preferably, the fragment of antibody keeps the ability of immunologic opsonin conjugated antigen.

As used herein, term " fused protein " is meant polypeptide, it comprises the aminoacid sequence of first protein or polypeptide or its fragment or its functional fragment or its analogue or derivative, and the aminoacid sequence of heterologous protein, polypeptide or peptide (that is second protein or polypeptide or its fragment, analogue or the derivative that, are different from described first protein or its fragment, analogue or derivative).In one embodiment, fused protein comprises prevention or the therapeutical agent that merges with heterologous protein, polypeptide or peptide.According to this embodiment, described heterologous protein, polypeptide or peptide can be or not be dissimilar prevention or therapeutical agents.

As used herein, term " gene expression pattern (pattern) ", " gene expression profile (profile) " can exchange use and comprise the RNA of two or more biomarkers of the present invention or the expression pattern of protein.For example, can also be called as " nucleic acid array express spectra " and constitute the hybridization of most nucleic acid probes on array of most target nucleic acid sequences and hybridization about the gene expression pattern or the pattern of RNA product, thus distinguish bladder cancer or early stage bladder cancer individual with non-bladder cancer individuality." gene expression pattern ", " gene expression profile " can also refer to the pattern corresponding to the protein abundance level of two or more biomarkers of the present invention, and it is to determine by any method of the described protein level of measurement well known in the art.For example, described pattern can be the mathematic(al) representation of described pattern, for example mathematical equation, vector etc.

As described herein, term " hybridize in " and " hybridization " are meant and the non-covalent association reaction of the sequence-specific of complementary nucleic acid for example reaction on array between target nucleic acid sequence and the nucleic acid member.

As used herein, term " immunoglobulin (Ig) " is meant the protein of being made up of immunoglobulin gene encoded polypeptides or its Fab basically one or more.Known immunoglobulin gene comprises κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region gene, and many immune globulin variable region genes.By at the variable region gene (about 110 amino acid) of NH2-end with at the κ or the λ constant region genes encoding total length immunoglobulin (Ig) " light chain " (about 25Kd or 214 amino acid) of COOH-end.Similarly by one of variable region gene (about 116 amino acid) and other aforementioned constant region gene (for example, γ, about 330 amino acid of encoding) coding total length immunoglobulin (Ig) " heavy chain " (about 50Kd or 446 amino acid).

As used herein, the term " associating " about treatment is meant that application surpasses a kind of treatment (for example, surpassing a kind of preventive and/or therapeutical agent).The application of term " associating " does not limit the order to object administering therapeutic (for example, preventing and/or treating agent).Can use second treatment to object (for example, second preventive or therapeutical agent) before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, before 8 weeks or 12 weeks), simultaneously or subsequently (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, after 8 weeks or 12 weeks) use first treatment (for example, first prevention or therapeutical agent).

As used herein, when using term " disease indicators " or " bladder cancer index ", be to represent the expression pattern of diagnosing bladder cancer and/or commitment bladder cancer and can comprise the individual pattern of suffering from bladder cancer and/or commitment bladder cancer possibility of increase with respect to expression pattern.

As used herein, " the in-line coding district " of term gene is meant the 5 ' district that is positioned at gene defined herein and the polynucleotide (two strands or strand) between the 3 ' district." in-line coding district " is not shorter than 8 Nucleotide length and can reaches 1000 Nucleotide or longer.Other of described " in-line coding district " may length include but not limited to 10,20,25,50,100,200,400 and 500 Nucleotide.5 ', 3 ' and inner area be not eclipsed and can but need not be successive, and can but do not need to add up to the total length of described corresponding gene.

As used herein, " the inner peptide zone " of term polypeptide is meant at the N-terminal district of polypeptide and the peptide sequence between the C-terminal district, as defined herein." the inner peptide zone " of polypeptide is not shorter than 3 amino acid longs and can reaches 350 amino acid or longer.Other of " the inner peptide zone " of polypeptide may length include but not limited to 5,10,20,25,50,100 and 200 amino acid.

The N-terminal of polypeptide, C-terminal and inner peptide zone be non-eclipsed and can but need not be successive, and can but do not need to add up to the total length of described corresponding polypeptide.

As used herein, when " separation " or " purifying " when being used for nucleic acid, the expression native sequences is removed or is synthesized in non-natural environment (for example, synthetic) from its normal cellular environment (for example, karyomit(e)).Therefore, " separation " or " purifying " sequence can be in cell-free solution or be placed in the different cellular environments.Term " purifying " does not mean that described sequence has only Nucleotide to exist, but does not have (about 90-95% is pure) natural non-nucleotide material coupled basically, and distinguishes mutually with isolating karyomit(e) thus.

As used herein, term " separation " and " purifying " are in the protein agent (for example, peptide, polypeptide, protein or antibody) category in be meant the protein agent, it does not have cellular material basically, and do not come from some embodiments, the heterologous protein agent (that is contaminating protein matter) of its derived cell or tissue basically, perhaps when using chemosynthesis, it does not have precursor or other chemicals basically.Phrase " acellular substantially material " comprises protein agent prepared product, and wherein said protein agent separates with the groups of cells phase-splitting of cell, described protein agent be from described cellular segregation or produce through reorganization.Therefore, do not have the protein agent of cellular material to comprise protein agent prepared product basically, it has less than the heterologous protein agent of about 30%, 20%, 10% or 5% (dry weight) (for example, protein, polypeptide, peptide or antibody; Also be called as " contaminating protein matter ").When described protein agent is when reorganization produces, also preferably do not contain the substratum of described protein agent substantially, that is, the substratum representative is less than the protein prepared product of about 20%, 10% or 5% volume.When producing described protein agent by chemosynthesis, preferably there are not precursor or other chemicals basically, that is, will be referred to described protein agent synthetic precursor or other chemicals separately.Therefore, such protein agent prepared product has precursor or the compound that is less than being different from of about 30%, 20%, 10%, 5% (dry weight) described proteins of interest matter agent.Preferably, protein agent disclosed herein is isolated.

As used herein, term " expression level " is meant with well known to a person skilled in the art the method for determining definite specific nucleic acid or proteinic amount corresponding to gene.For RNA, hnRNA, mRNA or mRNA splice variant corresponding to biomarker of the present invention, can determine expression level such as real-time quantitative RT PCR by hybridization and other method of masurement, described method of masurement comprises application

Green,

And molecular beacon (Molecular Beacons) technology.It should be noted that definite differential expression level used herein can comprise the difference between visual inspection appointment nucleic acid or the proteinic amount, for example by analyzing Northern trace or Western trace.

As used herein, " part " is molecule, and its specificity combination is by the polypeptide of the coded by said gene of biomarker of the present invention.Part can be nucleic acid (RNA or DNA), polypeptide, peptide or compound.Part of the present invention can be the peptide part, for example, and support peptide, linear peptides or cyclic peptide.In a preferred embodiment, described polypeptide ligand is an antibody.This antibody can be people's antibody, chimeric antibody, recombinant antibodies, humanized antibody, monoclonal antibody or its Fab or polyclonal antibody.Described antibody can be complete immunoglobulin (Ig), for example, and IgA, IgG, IgE, IgD, IgM or its hypotype.Described antibody can coupling function part (for example, compound with biology or chemical functional), its can be second kind not homopolypeptide (a second differentpolypeptide) but, medicine, cytotoxic agent test section or solid phase carrier (solidsupport).Polypeptide ligand of the present invention for example antibody and polypeptide interacts, and described polypeptide is coded by one of gene of biomarker, and described interaction has high-affinity and specificity.For example, described polypeptide ligand is in conjunction with polypeptide, and described polypeptide is coded by one of gene of biomarker, and wherein affinity costant is at least 10 ⁷M ^-1, preferably at least 10 ⁸M ^-1, 10 ⁹M ^-1Perhaps 10 ¹⁰M ^-1

As used herein, term " great majority " be meant expression surpass total composition 50% (for example, 51%, 60% or 70% or 80% or 90% or be up to 100%) amount.Term " great majority " when being used for array, its expression surpass the total nucleic acid member 50% (for example, 51%, 60% or 70% or 80% or 90% or be up to 100%), the solid phase substrate stable bond of described nucleic acid member and array.

As used herein, term " control " is meant the beneficial effect that comes from treatment (for example, preventing or therapeutical agent) that object obtains, and this treatment does not cause osteoarthritis to be cured.In certain embodiments, use one or more treatments with " control " osteoarthritis, thereby prevent the development or the deterioration of osteoarthritis to object.

As used herein, " mRNA integrity " is meant the quality from the mRNA extract of tissue sample or blood sample.In one embodiment, by with method well-known in the art (for example, by RNA agarose gel electrophoresis (for example, Ausubel et al., John Weley ﹠amp; Sons, Inc., 1997, Current Protocols in Molecular Biology)) detect the integrity whether RNA is degraded to determine mRNA.Preferably, the mRNA sample have good integrity (for example, the degraded of mRNA is less than 10%, preferably be less than 5% and more preferably less than 1%) therefrom extract the cartilage of mRNA or the gene expression dose of blood sample with representative truly.

As used herein, the patient with present available therapy for treating is described in term " unresponsive (non-responsive) " and " obstinate ", and one or more symptoms of its bladder cancer are not treated fully clinically and/or alleviated.

As used herein, term " normally " be meant do not show any bladder cancer sign or its symptom (comprising blood urine) and be not diagnosed as individuality or the population of individuals of suffering from bladder cancer or suffering from the bladder cancer possibility.Preferably, described " normally " is meant and suffers from individuality or the population of individuals that the bladder cancer risk does not have increase.In addition, preferred described normal individuality is not accepted to influence the pharmacological agent of bladder cancer and is not suffered from any other disease by diagnosis.More preferably, normal individual comparing with the sample of being checked has similar sex, age." normally " also represented from the isolating sample of normal individual and comprised from isolating total RNA of normal individual or mRNA according to the present invention.The sample that picks up from normal individual can comprise from the isolating RNA of tissue sample.

As used herein, " nucleic acid " and term " polynucleotide " is used interchangeably and its ordinary representation is any polybribonucleotide or polydeoxyribonucleotide, it can be not modified RNA or DNA or modified RNA or DNA or its any combination." nucleic acid " includes but not limited to single-and two-chain nucleic acid.As used herein, term " nucleic acid " also comprises aforesaid DNA or RNA, and it comprises the base of one or more modifications.Therefore, be " nucleic acid " for stability or other DNA or RNA former thereby that its skeleton has been carried out modifying.Term used herein " nucleic acid " comprises the nucleic acid form of modifying through chemistry, enzymatic or metabolism, and virus and the characteristic DNA of cell (for example comprising simple and complicated cell) and the chemical species of RNA." nucleic acid " or " nucleotide sequence " can also comprise that regional or its any combination and embodiments more according to the present invention of strand or double-stranded RNA or DNA can comprise expressed sequence tag note (EST).EST is the part (that is, " label " of sequence) of the sequence of gene through expressing, thereby its reverse transcription by the mRNA zone forms cDNA.

As herein defined, " nucleic acid array " is meant numerous unique nucleic acid (perhaps " nucleic acid member "), and it is attached to upholder, and wherein each nucleic acid member is attached in the particular pre-selected zone of upholder.In one embodiment, the nucleic acid member who is attached to support surface is DNA.In a preferred embodiment, the nucleic acid member who is attached to support surface is cDNA or oligonucleotide.In another preferred embodiment, the nucleic acid member who is attached to support surface is by polymerase chain reaction (PCR) synthetic cDNA.Term used herein " nucleic acid " can exchange with term " polynucleotide " and use.In another preferred embodiment, " nucleic acid array " is meant and is attached to Southern and/or the nitrocellulose filter of Northern engram technology use or numerous unique nucleic acids of other film.

As used herein, " nucleic acid probe " comprise can be on array the nucleic acid of complementary sequence by several non covalent bond binding interactions (comprising that the complementary base pairing interacts) bind nucleic acid member.As used herein, nucleic acid probe can be with comprising natural (that is, A, G, C or T) or the base (7-deazaguanine nucleosides, inosine etc.) of modifying.In addition, can connect the base in the nucleic acid probe by the mode of connection that is different from phosphodiester bond, if it does not disturb hybridization (that is, nucleic acid probe under the rigorous condition of standard or selective cross condition still specificity in conjunction with its complementary sequence).Therefore, nucleic acid probe can be a peptide nucleic acid(PNA), and wherein the base of Zu Chenging connects by peptide bond rather than phosphodiester bond.

As used herein, " nucleic acid target " or " nucleic acid member " be defined as can be in conjunction with the nucleic acid of array.Described nucleic acid target can be the isolated nucleic acid sequences corresponding to gene or its part, and perhaps described nucleic acid target can be total RNA or the mRNA from sample separation.Preferably, described nucleic acid target or nucleic acid marking are cartilage, blood or the synovia extracts that comes from the people.More preferably, described nucleic acid target is strand or double-stranded DNA, RNA or DNA RNA hybrid, and they come from total RNA extract of people's cartilage, blood or synovia, and preferably from the mRNA extract.

In one embodiment, the conventional nucleic acid array that is incorporated into ' target ' sequence of array can be the complete genomic representative of people, Affymetrix chip for example, and will comprise that separating bio tag application described gene of two or more tables 1 or gene probe or that be made up of them is in conventional arrays.

In another embodiment, the sequence that is incorporated into array can be isolating oligonucleotide, cDNA, EST or PCR product, and they are corresponding to the biomarker of the total cell RNA of the present invention that is applied to array.

As used herein, term " oligonucleotide " is defined as comprising two or more and preferably surpasses the molecule of three deoxyribonucleotides and/or ribonucleotide.Its accurately big young pathbreaker is depended on many factors, and these factors depend on the final function and the purposes of oligonucleotide.Described oligonucleotide can be about 8 long to about 1000 Nucleotide.Although can use the oligonucleotide of 8-100 Nucleotide in the present invention, preferred oligonucleotide scope about 8 is long to about 15 bases, about 8 long to about 20 bases, about 8 long to about 25 bases, about 8 long to about 30 bases, about 8 long or about 8 long to about 50 bases to about 40 bases.

As used herein, phrase " medicinal acceptable salt " includes but not limited to the salt of acidity or basic group, and it can be present in uses in the inventive method institute compounds identified.Basic cpd can form many kinds of salt with various inorganic and organic acids.The medicinal acid of accepting acid salt that can be used to prepare this basic cpd is those acid that form non-toxicity acid salt, be that described salt comprises the medicinal negatively charged ion of accepting, include but not limited to sulfuric acid, citric acid, toxilic acid, acetate, oxalic acid, hydrochloric acid, Hydrogen bromide, hydroiodic acid HI, nitric acid, sulfuric acid, bisulphate (bisulfate), phosphoric acid, acid phosphoric acid, Yi Yansuan salt (isonicotinate), acetate, lactic acid salt, salicylate, Citrate trianion, the acid Citrate trianion, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate salt, succinate, maleate, gentisate (gentisinate), fumarate, gluconate, saccharic acid salt (glucaronate), sucrose hydrochlorate (saccharate), formate, benzoate, glutaminate, mesylate (methanesulfonate), esilate (ethanesulfonate), benzene sulfonate (benzenesulfonate), tosilate and embonate are (promptly, 1,1 '-methylene radical-two-(2-hydroxyl-3-naphthoate)).The compound that comprises amino part can form medicinal acceptable salt with each seed amino acid (except that above-mentioned various acid).Acidic cpd can form alkali salt (base salts) with the various medicinal positively charged ions of accepting.The example of described salt comprises basic metal or alkaline earth salt, and especially calcium, magnesium, sodium, lithium, zinc, potassium and molysite.

As used herein, " polynucleotide " are contained and are surpassed 8 Nucleotide long double-stranded DNA, single stranded DNA and two strands or single stranded RNA.

As used herein, " by ... the encoded polypeptides sequence " be meant the aminoacid sequence that obtains after the protein coding region translation of gene, as herein defined.Discern corresponding peptide sequence by Genbank registration number (see Table 3 and/or table 6 and/or table 12) identification about the mRNA nucleotide sequence of each biomarker of the present invention and by protein registration number (see Table 3 and/or table 6 and/or table 12).

When with protein or protein fragments immunity host animal, proteinic many zones can inducing producing specificity in conjunction with the antibody of described protein designated area or three-dimensional structure; These zones or structure are called as epi-position or antigenic determinant.As used herein, " antigenicity fragment " is meant the some parts of polypeptide, and it contains one or more epi-positions.Epi-position can be linear, mainly comprises from described antigenic linear order, or conformation, comprise by other sequence with mode of inheritance separate but in the sequence of structurally getting together at the binding site of polypeptide ligand." antigenicity fragment " can be the random length of 5000,1000,500,400,300,200,100,50 or 25 or 20 or 10 or 5 amino acid longs at most.

As used herein, " preselected area (pre-selected region) ", " prospective region (predefined region) " or " unique location " are meant the regional area on the matrix, be intended to deposit nucleic acid with it, and be expressed as " institute's favored area " in the others of this paper alternatively or only be " zone ".Preselected area can have suitable shape arbitrarily, for example, and circle, rectangle, ellipse, wedge shape etc.In some embodiments, preselected area is less than about 1cm ², be more preferably less than 1mm ², still be more preferably less than 0.5mm ², and in some embodiments less than 0.1mm ²Nucleic acid member in " preselected area ", " prospective region " or " unique location " is a nucleic acid member that can determine its identity (for example, sequence) according to its position on described zone or unique location.

As used herein, term " prevention ", " preventing " are meant owing to use one or more compounds of identifying according to the inventive method or use development, recurrence or the outbreak that so combination of compound and another treatment causes preventing mild osteoarthritis or its one or more symptoms.

Term used herein " primer " is meant oligonucleotide, no matter it is the natural form that produces of the restriction digest with purifying or is produced by synthetic, when it places appropriate condition following time can be as the synthetic initiation site, the synthetic of primer extension product (it is complementary to nucleic acid chains) brought out under the described conditions, that is, exist Nucleotide and inductor such as archaeal dna polymerase and suitable temperature and pH condition under.Described primer can be strand or double-stranded and must sufficiently long, thereby starts the synthetic of expection extension products under the condition of described inductor existing.The exact length of described primer will depend on many factors, comprise temperature, primer source and method therefor.For example, for diagnostic use, according to the complicacy of target sequence, Oligonucleolide primers comprises 15-25 or more Nucleotide usually, although it can comprise Nucleotide still less.Relating to the factor of determining the primer suitable length is easy to know for those skilled in the art.Usually, the method for specifically described design of the present invention and selection primer is according to method standard and well-known in the art, sees Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S. (1995) General Concepts for PCR PrimerDesign.In:PCR Primer, A Laboratory Manual (Eds.Dieffenbach, C.W, and Dveksler, G.S.) Cold Spring Harbor Laboratory Press, New York, 133-155; Innis, M.A., and Gelfand, D.H. (1990) Optimization of PCRs.In:PCR protocols, A Guide to Methods and Applications (Eds.Innis, M.A., Gelfand, D.H., Sninsky, JJ., and White, T.J.) Academic Press, San Diego, 3-12; Sharrocks, A.D. (1994) The design of primers for PCR.In:PCRTechnology, Current Innovations (Eds.Griffin, H.G., and Griffin, A.M, Ed.) CRC Press, London, 5-11.

As used herein, term " probe " expression oligonucleotide and its analogue and be meant the chemical species of certain limit, it is by the described polynucleotide target sequence of identification that interacts of the hydrogen bonded with the nucleotide base of target sequence.Described probe or target sequence can be strand or double-stranded RNA or strand or the DNA of two strands or the combination of DNA and RNA base.At least 8 Nucleotide of probe are long and less than the total length of whole gene.Probe can be 10,20,30,50,75,100,150,200,250,400,500 and mostly to be 2000 Nucleotide most long.Probe can comprise modified oligonucleotide, can pass through the label that fluorescence, chemoluminescence etc. detect thereby have.Make it not only have detectable label but also have quencher molecule thereby can also modify described probe, for example

And Molecular

Probe.

Oligonucleotide and its analogue can be RNA or DNA, and perhaps the analogue of RNA or DNA is commonly called antisense oligomers or antisense oligonucleotide.Such RNA or DNA analogue include but not limited to the sugar-modified thing of 2-' O-alkyl, methylphosphonate, thiophosphatephosphorothioate, phosphorodithioate, formacetal, 3 '-thioformacetal, sulfone, sulfamate and nitroxide backbone modification thing and the adorned analogue of base portion.In addition, the oligomer analogue can be a polymer, wherein sugar moieties has been modified or has been fit to part by another and replaced, De polymer includes but not limited to morpholino analogue and peptide nucleic acid(PNA) (PNA) analogue (Egholm therefrom, et al.Peptide Nucleic Acids (PNA)-Oligonucleotide Analogues with an AchiralPeptide Backbone, (1992)).

Probe can also be any oligonucleotide analogs type mixture together or the mixture that makes up with n DNA or RNA.Simultaneously, described oligonucleotide and its analogue can use separately or be used in combination with one or more other oligonucleotide or its analogue.

As used herein, term " product of biomarker " or " biomarker product " are meant RNA or protein, it corresponding to described biomarker or by its coding (promptly, from gene or genetic elements is transcribed or from RNA translation, described RNA transcribes from described gene or genetic elements).For example, in some embodiments, the RNA that produces from described biomarker can comprise one or more following species: hnRNA, mRNA and/or one or more mRNA splice variants.In some embodiments, the protein that produces from molecule marker can be included in any protein of finding the blood, and described protein is corresponding to the RNA that produces from described biomarker.

As used herein, term " preventive " is meant any compound that can be used for preventing osteoarthritis.In certain embodiments, term " preventive " is meant compounds identified in screening assay method described herein.In certain embodiments, term " preventive " is meant reagent outside institute's authenticating compound in screening assay method described herein, and known described reagent can be used for or or at present be used for outbreak, generation and/or the development of prevention or prevention osteoarthritis or its one or more symptoms.

As used herein, phrase " prevention significant quantity " is meant that () amount for example, preventive, it is enough to cause preventing generation, recurrence or the outbreak of osteoarthritis or its one or more symptoms in treatment.

As used herein, term " protein " and " polypeptide " and " protein agent " can be exchanged use, and it is meant the amino acid chain that is connected together by peptide bond, and it can randomly comprise natural or non-natural amino acid.Randomly, described protein or peptide can comprise other molecule except that amino acid.Described chain can be a random length.In a specific embodiments, protein is by being less than 200, being less than 175, being less than 150, being less than 125, being less than 100, being less than 50, being less than 45, being less than 40, being less than 35, being less than 30, being less than 25, being less than 20, being less than 15, being less than 10 or be less than 5 amino acid and connect by peptide bond and form.In another embodiment, protein is connected by peptide bond by at least 200,250,300,350,400,450,500 or more amino acid form at least at least at least at least at least at least.

As used herein, " majority " or " one group " be meant and surpass 2, for example 3 or more, 10 or more, 100 or more or 1000 or more or 10000 or more.

As used herein, term " RNA part " and " its part " are meant the rna transcription thing in the category of the RNA of biomarker of the present invention product, it comprises at least 6, at least 9, at least 15, at least 18, at least 21, at least 24, at least 30, at least 60, at least 90, at least 99, at least 108 of the RNA product of biomarker of the present invention or the nucleotide sequence of polynucleotide more.

Term used herein " product of biomarker of the present invention " is meant by RNA and/or protein corresponding to the genetic expression of biomarker of the present invention." the RNA product of biomarker of the present invention " comprises one or more products, it can comprise all or some splice variant of heterogeneous nuclear RNA (" hnRNA "), mRNA and mRNA, measures according to its expression of instruction disclosed herein to be used as biomarker." protein of biomarker of the present invention " comprises the product of one or more described biomarkers, and it can comprise the modifying protein after protein, protein variant and any translation.

As used herein, term " selective amplification " or " selective amplification " are meant a kind of process, produce one or the particular target nucleotide sequence of multiple copied from the template nucleic acid selectivity thus.Selective amplification or selective amplification compare with common amplification, it can be used as with for example random primer and oligomerization dT primer are combined and comes amplifying nucleic acid sequence (for example, the mRNA) method of colony.Preferably implement selective amplification by polymerase chain reaction (Mullis and Faloona, 1987, Methods Enzymol.155:335).

As used herein, term " selective binding " is meant that in the protein category that the present invention is contained the specificity of any two peptides, protein, polypeptide and antibody interacts, wherein compare with antibody with any other peptide, protein, polypeptide, described interaction preferably takes place between any two peptides, protein, polypeptide and antibody.For example, when two molecules are protein molecule, structure on described first molecule identification and in conjunction with the structure on described second molecule, rather than other protein.Term used herein " selective binding " expression molecule is in conjunction with its specificity binding partner, and wherein said molecule is that at least 2 times of affinities and preferred at least 10 times, 20 times, 50 times, 100 times or the higher affinity when surpassing it in conjunction with non-specific molecules combines with its specificity binding partner.

" selective cross " used herein is meant in category of the present invention and occurs in polynucleotide and the RNA of biomarker of the present invention or the hybridization between its complement or the protein that the present invention comprises, wherein compare with the RNA product of other gene in the described genome, described hybridization makes described Nucleotide preferentially in conjunction with the RNA product of biomarker of the present invention.In a preferred embodiment, the polynucleotide of " selective cross " be with greater than 70%, greater than 80%, greater than 90% and most preferably 100% (promptly with other RNA material generation cross hybridization preferably less than 30%, less than 20%, less than 10%) the selectivity polynucleotide of hybridizing.The polynucleotide that it will be understood by those skilled in the art that the RNA product of " selective cross " biomarker of the present invention can be by considering length and forming to come definite.

As used herein, " specific hybrid ", " specific hybridization " be meant when two nucleotide sequences complementary substantially (for 14-25 nucleotide fragments at least, at least about 65% complementation, preferably at least about 75% complementation, more preferably at least about 90% complementation) time generation hybridization.See Kanehisa, M., 1984, Nucleic acids Res., 12:203, it incorporates this paper into by reference.Therefore, expection mispairing is to a certain degree allowed.Such mispairing can be little, such as single-, two-or three-individual Nucleotide.As an alternative, the mispairing zone can comprise ring (loop), and it is defined as in the zone that has mispairing in 4 of continuous series or a plurality of Nucleotide.The efficient and the selectivity of two nucleic acid hybridizations of many factor affecting, for example, the hybridization of nucleic acid member and target nucleic acid sequence on array.These factors comprise nucleic acid member's length, nucleotide sequence and/or composition, hybridization temperature, damping fluid composition and potential steric hindrance in the zone that the nucleic acid member need be hybridized.Between the annealing efficiency of length nucleic acid and nucleic acid and target sequence and accuracy, there is positive correlation.Specifically, the relatively shorter sequence of longer sequence has higher melting temperature(Tm) (T _M), and in specified target sequence, seldom may be repeated, non-specific hybridization is minimized.Hybridization temperature and nucleic acid member annealing efficiency inverse change.Similarly, the concentration and the annealing efficiency inverse change of organic solvent in hybridization mixture (for example methane amide) help annealing and increase salt concn in hybridization mixture.Under rigorous annealing conditions, the nucleic acid hybridization efficient that long nucleic acid is relatively lacked is higher, and it also is sufficient under the condition of relaxing more.

As used herein, term " specificity in conjunction with " is meant the interaction of two molecules, for example part and protein or peptide or antibody and protein or peptide, and the existence of ad hoc structure on each molecule is depended in wherein said interaction.For example, when described two molecules are protein molecule, in the identification of the structure on first molecule and in conjunction with the structure on second molecule, rather than general protein." specificity combination " used herein expression molecule is in conjunction with its specificity binding partner, it is that at least 2 times affinity and preferred at least 10 times, 20 times, 50 times, 100 times or higher affinity combines with its specificity binding partner when surpassing it in conjunction with non-specific molecules.

As used herein, term " the rigorous condition of standard " and " rigorous condition " expression has only when having 95% and preferably just hybridize during at least 97% consistence between the sequence at least, and wherein said consistence zone comprises at least 10 Nucleotide.In one embodiment, with sequence after 42 ℃ of overnight incubation, described sequence is hybridized under rigorous condition, uses rigorous lotion (0.2 * SSC, 65 ℃) afterwards.The rigorous degree of cleaning can change according to temperature, pH, ionic strength, divalent cation concentration, volume and the time length of cleaning.For example, can implement the preciseness that hybridization changes hybridization by the temperature under the probe melting temperature(Tm).The melting temperature(Tm) of probe can be used following formula and calculate:

To the oligonucleotide probe between 14-70 Nucleotide length, the centigradetemperature of melting temperature(Tm) (Tm) can be used following formula and calculate: Tm=81.5+16.6 (log[Na+])+0.41 (G+C mark)-(600/N), wherein N is the length of oligonucleotide.

For example, in the hybridization buffer with about 1M concentration Na+, hybridization temperature can drop to 42 ℃ from 68 ℃ with 5 ℃ increment.After the hybridization, can be with 2 * SSC under hybridization temperature, the 0.5%SDS cleaning filter membranes.These conditions are in " moderate preciseness " condition of being considered to more than 50 ℃, in being considered to below 50 ℃ " low preciseness condition "." moderate preciseness " hybridization conditions of a specific embodiment is a condition of implementing above-mentioned hybridization at 55 ℃." low preciseness " hybridization conditions of a specific embodiment is a condition of implementing above-mentioned hybridization at 45 ℃.

If implement hybridization in containing the solution of methane amide, the melting temperature(Tm) of annealing nucleic acid chains can be used following Equation for Calculating: Tm=81.5+16.6 (log[Na ⁺])+0.41 (G+C mark)-(0.63% methane amide)-(600/N), wherein N is the length of probe.

For example, can (in such as 6 * SSC), under 42 ℃ of conditions, implement hybridization containing the damping fluid of methane amide.In this case, the concentration of methane amide can drop to 0% to identify the clone of probe homology level reduction from 50% with 5% increment in hybridization buffer.After the hybridization, can be with 6 * SSC, 0.5%SDS is at 50 ℃ of cleaning filter membranes.When hybridization solution comprises methane amide more than 25%, think that hybridization conditions is " moderate preciseness " condition, when hybridization solution comprises methane amide below 25%, think " low preciseness " condition." moderate preciseness " hybridization conditions of a specific embodiment is to implement the condition of above-mentioned hybridization at 30% methane amide." low preciseness " hybridization conditions of a specific embodiment is to implement the condition of above-mentioned hybridization at 10% methane amide.

As used herein, term " object " and " patient " and " individuality " can exchange use, and they are meant animal (for example, Mammals, fish, Amphibians, Reptilia, bird and insect).In a specific embodiments, to as if Mammals (for example, inhuman Mammals and people).In another embodiment, to liking pet (for example, dog, cat, cavy, monkey and bird), domestic animal (for example, horse, ox, pig, goat and chicken) or laboratory animal (for example, mouse and rat).In another embodiment, to liking primate (for example, chimpanzee and people).In another embodiment, to liking the people.

As used herein, term " collaborative " is meant and uses one of method described herein compounds identified and another treatment (for example, reagent) combination, its additive effect than the difference treatment is more effective.Preferably, described other treatment or at present has been used for preventing, treat, control or improving bladder cancer or its symptom.The synergistic effect of each treatment (for example, preventive or therapeutical agent) combination allows to use described treatment to what the bladder cancer object was used one or more treatments than low dosage and/or with lower frequency.Utilization than the treatment of low dosage (for example, preventive or therapeutical agent) and/or reduced and use the relevant toxicity of described reagent to object with the ability that lower frequency is used described treatment, and do not reduce described treatment in the effect of preventing, treat, control or improving in the bladder cancer.In addition, synergistic effect can cause various treatments (for example, all ingredients) to improve in the effect of preventing, treat, control or improving in the bladder cancer.Deleterious or the undesirable side effect relevant with using arbitrary treatment separately can be avoided or reduce to the synergistic effect of various treatments at last, (for example, preventive or therapeutical agent) combination.

As used herein, term " therapeutical agent " is meant any compound, and it can be used to treatment, controls or improve bladder cancer or its one or more symptoms.

As used herein, term " treatment significant quantity " is meant is enough to cause bladder cancer or its one or more doing well,improvings, prevent the bladder cancer development, cause the bladder cancer degeneration or strengthen or (for example improve another treatment, the treatment of result of treatment therapeutical agent) (for example, therapeutical agent) amount.

As used herein, term " treatment " and " processing " (treat) are meant progress, seriousness and/or the recurrence that reduces or improve bladder cancer or its one or more symptoms, and this is by using one or more compounds of identifying according to the inventive method or using one or more compounds of identifying according to the present invention and the combination of another treatment produces.

As used herein, term " rise " or " expression level increase " relate to the product of genetic expression in category of the present invention, wherein can show according to using definite the measuring of described product of determination and analysis or other similar analysis, with comparing with the homologous genes the blood from normal individual or from individual isolating tissue with different evaluation morbid states or stage, from bladder cancer individuality or the evaluation morbid state or the individual isolating tissue and blood in stage according to the definite bladder cancer of AJCC sublevel guide (AJCC staging guidelines), described gene expression dose increases.According to the present invention, " expression level increase " is meant to express increases at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or greater than 1 times, be up to 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, 100 times or more, described expression be by, for example, measure according to the intensity for hybridization of the inventive method.For example, the sequence of rise comprises, and compares the sequence that increases at expression level from individual isolating tissue with bladder cancer or blood from the isolating tissue of normal individual.The sequence that raises can also comprise, compares the sequence that increases at expression level from individual isolating tissue with a certain stage bladder cancer or blood with the bladder cancer in another stage.

Description of drawings

At this present invention will be described in conjunction with the following drawings:

Fig. 1 is the synoptic diagram of one embodiment of the invention, and it shows that 7 kinds of biomarkers may make up for correlated all 21 kinds among the embodiment 9.The ROC that returns (logistic regression) for the logistic by various every kind of sorters that may combination results of biomarker indicates with blueness.For every kind of resulting sorter, indicate susceptibility with redness, wherein specificity is set to 90%.Specificity about every kind of sorter indicates with green, and wherein susceptibility is set to 90%.

Fig. 2 is the synoptic diagram of one embodiment of the invention, and it shows 2 kinds of correlated all 210 kinds possibilities of biomarker in 7 kinds of biomarkers pointing out among the embodiment 9.Indicate with blueness for the regressive ROC of logarithm by various every kind of sorters that may combination results of biomarker.For every kind of resulting sorter, indicate susceptibility with redness, wherein specificity is set to 90%.Specificity about every kind of sorter indicates with green, and wherein susceptibility is set to 90%.

Fig. 3 is the synoptic diagram of one embodiment of the invention, and it shows 3 kinds of correlated 1330 kinds of 1295 kinds possible (note that and can implement the limit search, but do not finish in this case) of biomarker in 7 kinds of biomarkers pointing out among the embodiment 9.Indicate with blueness for the regressive ROC of logarithm by various every kind of sorters that may combination results of biomarker.For every kind of resulting sorter, indicate susceptibility with redness, wherein specificity is set to 90%.Specificity about every kind of sorter indicates with green, and wherein susceptibility is set to 90%.

Fig. 4 is the synoptic diagram of one embodiment of the invention, and it shows in 7 kinds of biomarkers pointing out among the embodiment 9 correlated 5985 kinds possible 5250 kinds of 4 kinds of biomarkers (note that can by implementing the limit search analysis time that only increases computer).Indicate with blueness for the regressive ROC of logarithm by various every kind of sorters that may combination results of biomarker.For every kind of resulting sorter, indicate susceptibility with redness, wherein specificity is set to 90%.Specificity about every kind of sorter indicates with green, and wherein susceptibility is set to 90%.

3.2 embodiment

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, each described polynucleotide and biomarker selective cross.These biomarkers are genes identified in table 1.In one embodiment, can use at least two kinds each the expression level arbitrarily of these biomarkers that described composition measurement lists in table 1.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, each described polynucleotide and biomarker selective cross.These biomarkers are genes identified in table 1 and/or table 2.In one embodiment, can use at least two kinds each the expression level arbitrarily of these biomarkers that described composition measurement lists in table 1 and/or table 2.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, each described polynucleotide and biomarker selective cross.These biomarkers are genes identified in table 11.In one embodiment, can use at least two kinds each the expression level arbitrarily of these biomarkers that described composition measurement lists in table 11.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, and each described polynucleotide selective cross is to the RNA product of biomarker.In table 3, identified the RNA product of described biomarker.In one embodiment, can use at least two kinds the expression level that described composition is measured these RNA products.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, each described polynucleotide and biomarker selective cross.These biomarkers are genes identified in table 4.In one embodiment, can use at least two kinds the expression level that described composition is measured these biomarkers.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, each described polynucleotide and biomarker selective cross.Described biomarker is a genes identified in table 4 and table 5, and at least a table 4 that is selected from of these biomarkers.In one embodiment, can use at least two kinds the expression level that described composition is measured described biomarker.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described the set of two kinds or more of separation polynucleotide, and each described polynucleotide selective cross has wherein been identified the RNA product of described biomarker to the RNA product of biomarker in table 6.In one embodiment, can use at least two kinds the expression level that described composition is measured described RNA product.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, and each described polynucleotide selective cross is to biomarker.Described biomarker is a genes identified in table 7.In one embodiment, can use at least two kinds the expression level that described composition is measured described biomarker.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of separation polynucleotide, and each described polynucleotide selective cross is to biomarker.Described biomarker is a genes identified in table 10.In one embodiment, can use at least two kinds the expression level that described composition is measured described biomarker.In one embodiment, described polynucleotide compositions can be used for quantitative RT-PCR (QRT-PCR).

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, and the protein of every kind of isolated protein selective binding biomarker, wherein said biomarker are selected from the listed gene of table 1.In one embodiment, measure at least two kinds expression level of described biomarker with described composition.

This paper has described a kind of composition, it comprises the set of two kinds or more of isolated proteins, the protein of every kind of isolated protein selective binding biomarker, wherein said biomarker are the genes of listing in table 1 and table 2, and at least a biomarker is selected from table 1.In one embodiment, measure at least two kinds expression level of described biomarker with described composition.

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, and the protein of every kind of isolated protein selective binding biomarker, the protein of wherein said biomarker are the protein of listing in table 3.In one embodiment, measure at least two kinds expression level of described protein with described composition.

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, and the protein of every kind of isolated protein selective binding biomarker, wherein said biomarker are the genes of listing in table 4.In one embodiment, measure at least two kinds expression level of described biomarker with described composition.

This paper has described a kind of composition, it comprises the set of two kinds or more of isolated proteins, the protein of every kind of isolated protein selective binding biomarker, wherein said biomarker is selected from the gene of listing in table 4 and table 5, and at least a described biomarker is selected from table 4.In one embodiment, measure at least two kinds expression level of described biomarker with described composition.

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, the protein of every kind of isolated protein selective binding biomarker, and the protein of wherein said biomarker is selected from the protein of listing in table 6.In one embodiment, measure at least two kinds expression level of described protein with described composition.

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, the protein of every kind of isolated protein selective binding biomarker, and wherein said biomarker is selected from the gene of listing in table 7.In one embodiment, measure at least two kinds expression level of these biomarkers with described composition.

This paper has described a kind of composition, and it comprises the set of two kinds or more of isolated proteins, the protein of every kind of isolated protein selective binding biomarker, and wherein said biomarker comprises the gene of listing in table 10.In one embodiment, measure at least two kinds expression level of described biomarker with described composition.

In the composition of describing in the paragraph on this paper, described isolated protein is the part of the protein of biomarker, and these parts can be antibody, comprises monoclonal antibody.

In the composition of describing in the paragraph on this paper, described isolating polynucleotide can be strand or double-stranded RNA or strand or double-stranded DNA.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, wherein said one or more rna transcription things corresponding to one or more biomarkers of table 1 and

B) with one or more rna transcription things described in the blood of step a) each level with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at two or more rna transcription things of from the blood that individuality obtains, expressing, wherein said two or more rna transcription things are corresponding to one or more biomarkers of table 1 and table 2, wherein at least a described rna transcription thing corresponding to the biomarker of table 1 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, wherein said one or more rna transcription things corresponding to one or more biomarkers of table 4 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at two or more rna transcription things of from the blood that individuality obtains, expressing, wherein said two or more rna transcription things are corresponding to one or more biomarkers of table 4 and table 5, wherein at least a described rna transcription thing corresponding to the biomarker of table 4 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, wherein said one or more rna transcription things corresponding to one or more biomarkers of table 7 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, wherein said one or more rna transcription things corresponding to one or more biomarkers of table 10 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

Each the differential expression that wherein detects at one or more rna transcription things described in the comparison of step b) is the index of bladder cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, each of wherein said one or more rna transcription things corresponding to the biomarker that is selected from table 1 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood with bladder cancer individuality,

The level of each of described one or more rna transcription things that c) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

D) whether the level of determining step described one or more rna transcription things a) can come the described transcript level of step b) is classified than the described transcript level of step c),

Wherein saidly determine that the step a) individuality has the index of bladder cancer.

This paper has described a kind of method of diagnosing or predicting individual commitment bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, each of wherein said one or more rna transcription things corresponding to the biomarker that is selected from table 4 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood with commitment bladder cancer individuality,

The level of each of described one or more rna transcription things that c) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

D) whether the level of determining step described one or more rna transcription things a) can come the described transcript level of step b) is classified than the described transcript level of step c),

Wherein saidly determine that the step a) individuality has the index of commitment bladder cancer.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, each of wherein said one or more rna transcription things corresponding to the biomarker that is selected from table 7 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood with bladder cancer individuality,

The level of each of described one or more rna transcription things that c) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

D) whether the level of determining step described one or more rna transcription things a) can come the described transcript level of step b) is classified than the described transcript level of step c),

Wherein saidly determine that the step a) individuality has the index of bladder cancer.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine level at one or more rna transcription things of from the blood that individuality obtains, expressing, each of wherein said one or more rna transcription things corresponding to the biomarker that is selected from table 10 and

The level of each of one or more rna transcription things that b) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood with bladder cancer individuality,

The level of each of described one or more rna transcription things that c) will be in the blood of step a) with compare in each level from described one or more rna transcription things in one or more blood that do not have a bladder cancer individuality,

D) whether the level of determining step described one or more rna transcription things a) can come the described transcript level of step b) is classified than the described transcript level of step c),

Wherein saidly determine that the step a) individuality has the index of bladder cancer.

This paper has described certain methods, comprises the certain methods of how diagnosing and/or predicting bladder cancer, and wherein blood sample is made up of cleaved whole blood, drop of blood or whole blood or droplets of whole blood.

This paper has described certain methods, comprises the certain methods of how diagnosing and/or predicting bladder cancer, and it also comprises from the step of described blood sample isolation of RNA.

This paper has described certain methods, comprises the method for how diagnosing and/or predicting bladder cancer, and it comprises each the step of level of determining one or more rna transcription things, and this can comprise quantitative RT-PCR (QRT-PCR).

In some cases, QRT-PCR uses the primer of hybridizing with described one or more transcripts or its complement.Described primer comprises those primers that 15-25 Nucleotide is long.

This paper has described certain methods, comprise the certain methods of how diagnosing and/or predicting bladder cancer, it comprises the step of each level of determining described one or more rna transcription things, this comprises first kind of volume isolated nucleic acid molecule of hybridization, and described isolated nucleic acid molecule is corresponding to described one or more transcripts, corresponding to the array that comprises second kind of volume isolated nucleic acid molecule.In these methods, described first kind of volume isolated nucleic acid molecule comprises RNA, DNA, cDNA, PCR product or EST.

This paper has described a kind of test kit that is used to diagnose or predict bladder cancer, and it comprises:

A) two kinds of biomarker specificitys cause instrument, and it is designed to produce to be complementary to be selected from table 1,4,7 or 10; The double-stranded DNA of 11 or 13 biomarkers in any, the wherein said first initiation instrument comprises the sequence of RNA, the cDNA of alternative hybridization and described biomarker complementation or EST to generate extension products, and the second initiation instrument, it can the described extension products of selective cross

B) have the enzyme of reverse transcriptase activity,

C) have the thermostability dna polymerase activity enzyme and

D) marking tool;

Wherein use the quantitative expression level that described primer detects biomarker described in institute's checked object.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine one or more proteinic levels of expressing from the blood that individuality obtains, wherein said one or more protein by one or more biomarkers of listing in table 1 coded and

The level of each of described one or more protein that b) will be in the blood of step a) with compare in each level from described one or more protein in one or more blood that do not have a liver cancer individuality,

Wherein detect comparison step b) described in the difference of each level of one or more protein are indexs of liver cancer in the step a) individuality.

This paper has described a kind of method of diagnosing or predicting individual bladder cancer, and it may further comprise the steps:

A) determine one or more proteinic levels of expressing from the blood that individuality obtains, wherein said one or more protein by one or more biomarkers of listing in table 1 coded and

The level of each of described one or more protein that b) will be in the blood of step a) with compare in each level from described one or more protein in one or more blood with bladder cancer individuality,

C) with described one or more protein in the step a) blood each level with compare in each level from described one or more protein in one or more blood that do not have a bladder cancer individuality,

D) whether determining step described one or more proteinic levels a) can classify to proteinic level described in the step b) than proteinic level described in the step c),

Wherein saidly determine that the step a) individuality has the index of bladder cancer.

This paper has described certain methods, comprise the method that is used to diagnose or predict bladder cancer, it comprises the step of each level of determining described one or more protein, this step comprises uses one or more antibody, and the protein of each his-and-hers watches 3 listed biomarker of wherein said one or more antibody has specificity.

This paper has described certain methods, comprises the method that is used to diagnose or predict bladder cancer, and it comprises one or more antibody, and described antibody can be monoclonal antibody, fv, scfv, dab, fd, fab and fab ' ₂

This paper has described the method for some identification of organism marks and/or biomarker combination, it can be used for prognostic analysis (prognosing) and/or diagnosing bladder cancer, comprise with the expression level of known biomarker with bladder cancer individuality and knownly do not have the described level of bladder cancer individuality to compare that described biomarker is selected from table 1 and/or table 2 or table 7 or table 10 or table 11 and/or table 12.

This paper has described the method for some identification of organism marks and/or biomarker combination, it can be used for prognostic analysis and/or diagnosis commitment bladder cancer, comprise with the expression level of known biomarker with commitment bladder cancer individuality and knownly do not have the described level of bladder cancer individuality to compare that described biomarker is selected from table 3 and/or table 4.

4. show

With reference to table 1-7 purpose that the present invention may be better understood and feature, it is included in after the embodiment part of specification sheets.

In one embodiment of the invention, table 1 is the table that shows biomarker, and described biomarker is distinguished bladder cancer and normal situation.With their genetic identifier (gene ID) note biomarker.Row 1 are AffySpotID, and row 2 are p values, and row 3 are that multiple changes (bladder cancer/contrast), and row 4 are GeneID, and row 5 are that gene symbol and row 6 are gene explanations.

In one embodiment of the invention, table 2 is the tables that show biomarker, and described biomarker is distinguished bladder cancer and normal situation, as identifying in PCT patent application PCT/US04/020836 before.With their gene I note biomarker.Row 1 are gene I, and row 2 are people RNA registration numbers, and row 3 are human protein registration numbers, and row 4 are that gene symbol and row 5 are gene explanations.

In one embodiment, table 3 is the tables that show corresponding to the product of the biomarker of identifying in the table 1, comprises the RNA product, and its reference registration number by nucleic acid is represented, and protein, and it is represented with reference to registration number by proteinic.With their gene I note and definite gene.Row 1 are AffySpotID, and row 2 are gene I, and row 3 are people RNA registration numbers, and row 4 are human protein registration numbers, and row 5 are that gene symbol and row 6 are gene explanations.

In one embodiment of the invention, table 4 is the tables that show the biomarker of distinguishing commitment bladder cancer and normal conditions.With their gene I note biomarker.Row 1 are AffySpotID, and row 2 are gene I, and row 3 are gene symbols, and row 4 are people RNA registration numbers, and row 5 are that human protein registration number and row 6 are gene explanations.

In one embodiment of the invention, table 5 is the tables that show the biomarker of distinguishing commitment bladder cancer and normal conditions, as identifying in PCT patent application PCT/US04/020836 before.With their gene I note biomarker.Row 1 are gene I, and row 2 are people RNA registration numbers, and row 3 are human protein registration numbers, and row 4 are that gene symbol and row 5 are gene explanations.

In one embodiment, table 6 is the tables that show corresponding to the product of the biomarker of identifying in the table 4, comprises the RNA product, and its reference registration number by nucleic acid is represented, and protein, and it is represented with reference to registration number by proteinic.With their gene I note and definite gene.Row 1 are AffySpotID, and row 2 are gene I, and row 3 are people RNA registration numbers, and row 4 are that human protein registration number and row 5 are gene symbols.

In one embodiment, table 7 is tables of the total biomarker of indicator gauge 1 and table 4.Row 1 are AffySpotID, and row 2 are p values, and row 3 are gene I, and row 4 are that gene symbol, row 5 are that people RNA registration number and row 5 are protein registration numbers.

Table 8 is the tables that exemplify data, and by expression data being applied to use the mathematical model of decilog (logit), described data can be used to form the ROC curve.

Table 9 is tables of enumerating the characteristic of possible reporter gene and reporter gene product.

In one embodiment, table 10 be show identify in the concrete option table 1 and in table 3 the further table of the biomarker of note.

In one embodiment, table 11 is the tables that show the selected works of same 349 kinds of biomarkers identifying in table 1, and with respect to testicular cell cancer or renal cell carcinoma, described biomarker is distinguished bladder cancer.With their gene I note biomarker.Row 1 are AffySpotID, row 2 are ReprRNA registration numbers, row 3 are gene I, row 4 are gene symbols, row 5 are that multiple changes (bladder cancer/contrast), row 6 are the adjustment to disease p value (bladder cancer/contrast) (BC is to C), and row 7 are that multiple variation (bladder cancer is to other cancer) and row 8 are the adjustment to bladder cancer p value (bladder cancer is to other cancer).

In one embodiment, table 12 is the tables that show the selected product of the biomarker of identifying corresponding to table 11, comprises the RNA product, and its reference registration number by nucleic acid is represented, and protein, and it is represented with reference to registration number by proteinic.With their gene I note and identified gene.Row 1 are AffySpoptID, and row 2 are gene I, and row 3 are gene symbols, and row 4 are that people RNA registration number and row 5 are human protein registration numbers.

In one embodiment, table 13 is tables of the concrete selected works of the biomarker that shows that his-and-hers watches 1 are identified.

Table 14 is the tables that show corresponding to the registration number of the sequence of the people RNA product of the selected biomarker of table 13 and human protein product.

Table 15 is that it is used according to embodiment 4 to the selected works at the biomarker Auele Specific Primer of the listed biomarker of table 13.

Table 16 shows, according to the description of embodiment 4, about the result of the real-time quantitative RT-PCR (QRT-PCR) of the RNA product of listing in table 13 biomarker.

In one embodiment of the invention, table 17 is the demonstrations to isolated antibody, the protein of the listed biomarker of described antibody selective binding table 13 and can obtaining from commerce.

In one embodiment of the invention, table 18 is selected works of biomarker Auele Specific Primer and corresponding biomarker specific probe, the double-stranded DNA of RNA product shown in their selective amplifications are complementary to (it is the product of biomarker shown in the table 13).

In one embodiment of the invention, table 19 shows the combination of selected 2 genes, and it can be used to screen bladder cancer.

Table 20 shows the classification set that utilizes 4 kinds of biomarker contrast combinations of one embodiment of the invention, and wherein said biomarker is selected from those biomarkers of table 13.

Table 21 shows the classification set that utilizes 3 kinds of biomarker contrast combinations of one embodiment of the invention, and wherein said biomarker is selected from those biomarkers of table 13.

Table 22 shows the classification set that utilizes 2 kinds of biomarker contrast combinations of one embodiment of the invention, and wherein said biomarker is selected from those biomarkers of table 13.

Table 23 shows the classification set that utilizes biomarker contrast combination of one embodiment of the invention, and wherein said biomarker is selected from those biomarkers of table 13.

5. detailed description of the invention

The part land productivity is with molecular biological routine techniques, microbiology and recombinant DNA technology in the present invention practice, and it is in the technical scope of this area.These technology have sufficient description in the literature.For example see Sambrook, Fritsch ﹠amp; Maniatis, 1989, Molecular Cloning:A Laboratory Manual,Second Edition; Oligonucleotide Synthesis(M.J.Gait, ed., 1984); Nucleic Acid Hybridization(B.D.Harnes ﹠amp; S.J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning(B.Perbal, 1984); And aseries, Methods in Enzymology(Academic Press, Inc.); Short Protocols In Molecular Biology, (Ausubel et al., ed., 1995).All patents, patent application and publication that this paper mentions, not only comprised above but also comprise followingly, their full content is by with reference to incorporating this paper into.

Invention disclosed herein has been identified biomarker and the biomarker combination that can be used for diagnosing bladder cancer and/or commitment bladder cancer.In order to use these biomarkers, the present invention has instructed the evaluation to the product of these biomarkers, and described product comprises RNA product and protein.Oligonucleotide, cDNA, DNA, RNA, PCR product, synthetic DNA, synthetic RNA and its fragment are also contained in the present invention, and perhaps specificity and/or selective cross are to other combination of the natural modified nucleotide of the RNA product of biomarker of the present invention.The present invention also discloses protein, peptide, antibody, part and its fragment, comprises Fab, and their specificitys and/or selective cross are to the protein of biomarker of the present invention.Can use RNA product to biomarker of the present invention have specificity and/or optionally those polynucleotide implement measurement that the RNA product of biomarker of the present invention and biomarker combination is expressed, thereby the expression of quantitative described RNA product.In a specific embodiments of the present invention, for described RNA product have specificity and/or optionally polynucleotide be probe or primer.In one embodiment, these polynucleotide are forms of nucleic acid probe, its can with the synthetical hybridization array.In another embodiment, which assortment of genes expression and the present invention that can use the described RNA product of commodity array measurement instructs be used for analyzing.In another embodiment, in some technology of using, such as real-time quantitative RT PCR, application examples as

Green or application

Perhaps Molecular Beacon technology, use RNA product to biomarker of the present invention with the form of probe and primer and have specificity and/or polynucleotide optionally, wherein use applied polynucleotide with the form of the probe of the probe of forward primer, reverse primer, TaqMan mark or Molecular Beacon mark.In a specific embodiments, the result who obtains from measure RNA product expression level of the present invention can import in the model of the present invention, and it is used to the combination of identification of organism mark, to determine the diagnostic result by this model definition.In a preferred embodiment, same method is used to produce expression data, and these data are used to produce mathematical model, and this model is used to diagnose the individuality of being checked.

The present invention also considers to use protein disclosed herein or polypeptide, and those skilled in the art will know that protein how to measure biomarker of the present invention.Can use the level of the technology of well known to a person skilled in the art (for example, described technology comprises such as Western trace, immunoprecipitation, protein microarray analysis etc.) measurement subsequently corresponding to the protein of biomarker of the present invention.Those skilled in the art should be understood that, the expression level of measuring the protein of biomarker of the present invention needs the protein of specificity or one or more proteins of selective binding, and described protein is corresponding to various biomarkers of the present invention.Can will represent in the data input model of protein expression level of biomarker of the present invention subsequently, determine by the defined diagnostic result of this model thereby the described model of generation is used for identifying described combination.In a preferred embodiment, the generation expression data that should use the same method produces the mathematical model that is used for diagnosing the individuality of checking with these data.

5.1 be used for sample of the present invention

Unless this paper points out in addition, can the method according to this invention use bladder body sample from object, can be blood sample, serum sample and lymphoglandula sample such as described sample.Can the method according to this invention obtain and the object sample that utilizes includes but not limited to asymptomatic object from object, manifest or show 1 of bladder cancer, 2,3, the object of 4 kinds or more symptoms, the object that has bladder cancer and/or commitment bladder cancer through clinical diagnosis, easily the object of trouble bladder cancer (for example, object with family's bladder cancer history, object and its mode of life with bladder cancer genetic predisposition cause them easily to suffer from the object of bladder cancer or increase generation bladder cancer possibility), suspect object with bladder cancer, stand the object of bladder associated conditions treatment, object with bladder cancer and at least a other illness (for example, have 2,3,4, the object of 5 kinds or more illnesss), do not stand the object of bladder cancer treatment, through the doctor (for example, the physician) is defined as healthy or do not have bladder cancer (that is, normal) object, bladder cancer had been carried out the object (comprise bladder cancer was carried out the object that success is treated) of treatment, the object of positive regulation bladder cancer and also do not suffered from the object of bladder cancer by diagnosis.

In another embodiment, can have the commitment bladder cancer from its object that obtains sample and utilization.In further embodiment, can from its obtain sample to as if the individuality that accepts inspection, wherein do not understand this individuality and whether have bladder cancer, and/or do not understand institute's checked object and have the bladder cancer in what stage.

5.1.1 tissue

In one aspect, use any known method and obtain tissue sample from one or more objects.In a specific embodiments, the individuality of suffering from bladder cancer from suspection obtains tissue.Preferably, before application, described tissue sample is kept in the liquid nitrogen.In a specific embodiments, the tissue sample that separates minimum 0.05g is to obtain total RNA.In another embodiment, separate the tissue sample of minimum 0.025g to obtain total RNA.The amount of the tissue sample that can use according to the present invention is enough to be used for detecting one or more nucleotide sequences of the present invention or aminoacid sequence.

5.1.2 blood

In one aspect of the invention, obtain blood sample according to method well-known in the art from object.Can obtain blood sample from any object described herein.In some embodiments, collect drop of blood by acupuncture subject's skin easily.As disclosed herein, drop of blood comprises the volume less than 1ml.In some embodiments, the drop of blood of collecting by acupuncture is unusual ideal.Can use method well known in the art (method of phlebotomize bloodletting especially well known in the art) and get blood from any part of subject's body (for example, refer to, hand, wrist, arm, leg, foot, ankle, stomach and neck).

Collected blood volume will depend on the level of comfort of collecting position, the necessary amount of the inventive method and object and change.Yet the advantage of one embodiment of the invention is that the essential blood volume of enforcement the inventive method is so little, to such an extent as to do not need more intervention property process in order to obtain sample.For example, in some embodiments, needed only is drop of blood.Can pass through, for example, acupuncture easily obtains described drop of blood.In some embodiments, the blood volume of collection is enough to detect a kind of, two kinds, three kinds, four kinds, five kinds, ten kinds or more kinds of expression of gene in arbitrary gene of listing in table 1-7 or 10-12.Similarly, in some embodiments, the blood volume of collection is to be less than or equal to 1ml, to be less than or equal to 500 μ l, to be less than or equal to 250 μ l, to be less than or equal to 200 μ l, to be less than or equal to 100 μ l, to be less than or equal to 50 μ l, to be less than or equal to 20 μ l, to be less than or equal to 10 μ l, to be less than or equal to 1 μ l, to be less than or equal to 0.5 μ l, to be less than or equal to 0.1 μ l or to be less than or equal to 0.01 μ l.Yet the present invention is not limited to described embodiment.In some embodiments, can use more blood, and in some embodiments, can use more blood and realize method of the present invention.Similarly, in different specific embodiments, collect 0.001ml, 0.005ml, 0.01ml, 0.05ml, 0.1ml, 0.15ml, 0.2ml, 0.25ml, 0.5ml, 0.75ml, 1ml, 1.5ml, 2ml, 3ml, 4ml, 5ml, 10ml, 15ml or more blood from object.In another embodiment, collect 0.001ml to 15ml, 0.01ml to 10ml, 0.1ml to 10ml, 0.1ml to 5ml, 1 to 5ml blood from object.

In some embodiments of the present invention, blood is stored in the K3/EDTA pipe.In another embodiment, can use the pipe that contains stablizer that is used to preserve blood, as U.S. Patent No. 6,617, disclosed in 170 (they incorporate this paper into by reference).In another embodiment, can use by PreAnalytiX the PAXgenen that Qiagen/BD company provides ^TMBlood RNA (the PAXgene of system ^TMBlood RNA system) collects blood.Still in another embodiment, can use the Tempus that provides by Applied Biosystems ^TMBlood RNA collection tube.Tempus ^TMCollection tube provides the closure plastics tubing of finding time, and it comprises the RNA stablizer that is used for whole blood collection.

Collected collection blood is before the method according to this invention is used, by randomly but preferably be kept under the refrigerating temperature, such as 4 ℃ or on ice.In some embodiments, described blood is maintained at 4 ℃ or at only 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours or maximum 24 hours on ice.In some embodiments, except preservation or the preservation of replacement, preserve isolating nucleic acid or protein for some time and use to treat the back to blood to blood.Can preserve described biomarker one hour or longer, one day or longer, all or longer, one month or longer, 1 year or a longer or indefinitely.

In one aspect, according to phlebotomize bloodletting method well-known in the art, from normal individuality or from being suffered from by diagnosis or suspecting that the individuality with osteoarthritis obtains whole blood.Whole blood comprises unassorted whole blood, the blood that it comprises the blood of having removed serum or blood plasma and separated RNA or mRNA according to method well-known in the art (for example, preferably under 300 to 800 * g conditions appropriateness centrifugal 5 to 10 minutes) from remaining blood sample.In a specific embodiments, unassorted whole blood is also contained, according to method well known in the art, with blood and lysis buffer (for example, lysis buffer (IL): 0.6g EDTA, 1.0g KHCO ₂, 8.2g NH ₄Cl regulates pH to 7.4 with NaOH) whole blood of combination treatment, centrifugal described sample also keeps cell precipitation, and extracts RNA or mRNA (for example, " cracked whole blood ") (seeing for example Sambrook et al.).It is preferred using whole blood because it avoided expensively with separate consuming timely hemorrhage in each cell type (Kimoto, 1998, Mol.Gen.Genet 258:233-239; Chelly J et al., 1989, Proc.Nat.Acad.Sci.USA 86:2617-2621; Chelly J et al., 1988, needs Nature333:858-860).

In some embodiments of the present invention, the whole blood of collecting from object is carried out classification (that is, being divided into different compositions).In specific embodiments more of the present invention, use technology well known in the art, from collecting washed corpuscles from the whole blood of object.For example, the whole blood that can use Ficoll-Hypaque (Pharmacia) gradient centrifugation to handle to collect from object is with washed corpuscles.Described centrifugal red corpuscle (red blood corpuscle) and various types of karyocytes and blood plasma are separated.Especially, the Ficoll-Hypaque gradient centrifugation can be used to separate peripheral blood leucocyte (PBL), but its method according to this invention is used.

By way of example but unrestriced mode can obtain scavenger cell according to the following stated.Extract blood by syringe, then through the Ficoll-Hypaque gradient centrifugation and from the peripheral blood separating monocytic cell of object.Be organized culture dish and under 37 ℃, hatched 1 hour with the serum of object oneself or with the pre-bag of AB+ human serum.Remove the cell that does not adhere to transfer pipet.The 1mM EDTA that cold (4 ℃) are dissolved in phosphate buffer soln is added to the attached cell that is present in culture dish and described culture dish was placed room temperature following 15 minutes.Collecting cell is with the RPMI buffer solution for cleaning and be suspended in the RPMI damping fluid.By scavenger cell is hatched at 37 ℃ with macrophage colony stimulating factor (M-CSF), can cause scavenger cell quantity to increase.Antibody from the anti-scavenger cell specific surfaces of mark mark (such as Mac-1) to described molecule coupling affinity compound that can be by coming with promote to the detection of scavenger cell with separate.Adaptable affinity compound includes but not limited to vitamin H, photobiotin, fluorescein isothiocyanate (FITC) or phycoerythrin (PE), other compound perhaps known in the art.Subsequently, by means commonly known in the art, be such as but not limited to various cell sorting methods, affinity chromatography and elutriator, will have adhered to the cell of traget antibody and separate with the cell that does not combine described antibody.

Using fluorescence-activated cell sorter (FACS) can the sorting hemocyte.Fluorescence-activated cell sorting (FACS) is a known method, and its fluorescent characteristic based on particle is come separate particle, comprises cell.See, for example, Kamarch, 1987, Methods Enzymol 151:150-165.Fluorescence in the laser excitation particle individuality partly causes producing little electric charge, and this allows electromagnetic separation positive and negative particle from mixture.With fluorescence dye (such as FITC or phycoerythrin) traget antibody or part, described antibody or part are to be used for detecting being present in the lip-deep blood cell antigen determinant of specific hemocyte.Described cell is hatched for some time with fluorescently-labeled antibody or part, and this section period is enough to allow the antibody of described mark or part in conjunction with cell.Allow described cell through cell sorter, this permission separates interested cell and other cell.The particle of FACS sorting can directly be deposited in each hole of microtiter plate and separate with auxiliary.

In some embodiments of the present invention, can also use the magnetic bead washed corpuscles.For example, can applied magnetic active cells sorting (MACS) technology sorting hemocyte, this technology is based on the method for particle in conjunction with the ability separating particle of magnetic bead (0.5-100m diameter).Can implement many kinds of available to magnetic microsphere and modify, comprise that covalency adds specific recognition cell-solid phase surface molecule or haptenic antibody.Subsequently, apply magnetic field, thereby operate selected pearl in the physics mode.In a specific embodiments, will be at the antibody coupling of hemocyte surface markers to magnetic bead.Subsequently described pearl and the mixing of hemocyte culture are combined with permission.Next allow cell pass through magnetic field, thereby isolate interested cell with hemocyte surface markers.Can separate these cells subsequently.

In some embodiments, can use antibody sandwich culture dish surface, and be used to according to the method washed corpuscles that is called elutriator.Can be with the separated ware of specific hemocyte specific antibody bag.At first cell can be added in the vessel with interested hemocyte specific antibody bag quilt.After thorough rinsing, the cell that is incorporated into these vessel will be the cell of expressing interested hemocyte mark.The example of cell-surface antigens determinant or mark comprises, but be not limited to the CD68 of the CD21 of the CD19 of the lymphocytic CD3 of the CD2 of T lymphocyte and natural killer cell, T, the lymphocytic CD28 of leukocytic CD11a, T, bone-marrow-derived lymphocyte, the CD20 of bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte, CD22, the CD23 of bone-marrow-derived lymphocyte, leukocytic CD29, monocytic CD14, hematoblastic CD41, hematoblastic CD61, granulocytic CD66, granulocytic CD67 and the monocyte and the scavenger cell of bone-marrow-derived lymphocyte.

Separation of whole blood can be become various types of cells,, and can the method according to this invention use the cell of these types such as white corpuscle, thrombocyte, red corpuscle etc.Can also the application standard technology divide myeloblast and agranulocyte and these cells the method according to this invention to use white corpuscle.Can the application standard technology granulocyte being divided into different cell types can the method according to this invention use such as neutrophilic granulocyte, oxyphie and basophilic leukocyte and these cells.Can the application standard technology divide lymphocytoblast (for example, T lymphocyte and bone-marrow-derived lymphocyte) and monocyte and these cells the method according to this invention to use agranulocyte.The application standard technology T lymphocyte and bone-marrow-derived lymphocyte can be separated and helper cell and cytotoxic T cell separated and these cells can the method according to this invention be used.Isolating hemocyte (for example, white corpuscle) is being used for before present method, can be with standard technique with described cell freezing.

The amount that can be used for blood sample of the present invention should enough detect one or more nucleic acid of the present invention or aminoacid sequence.In a specific embodiments, the scope that can be used for blood sample amount of the present invention is from 1 μ l to 100ml, preferred 10 μ l to 50ml, more preferably 10 μ l to 25ml and most preferably 10 μ l to 1ml.

5.1.3 serum

In one aspect of the invention,, at first separate blood sample from object according to method well-known in the art, this sample of centrifugation subsequently, thus serum and all the other components of blood are separated, obtain the serum of object thus.Can from any object described herein, obtain serum sample.The serum amount of collecting will change according to the level of comfort of the necessary amounts of the position of collection blood, the inventive method and object.Yet the advantage of one embodiment of the invention is, can be not need more intervention procedure in order to obtain sample so for a short time to such an extent as to the essential blood volume of implementing the enough serum of the inventive method is provided.For example, in some embodiments, needed is to bleed and the serum amount of collecting is to be equal to or less than 1ml, to be equal to or less than 500 μ l, to be equal to or less than 250 μ l, to be equal to or less than 200 μ l, to be equal to or less than 100 μ l, to be equal to or less than 50 μ l, to be equal to or less than 20 μ l, to be equal to or less than 10 μ l, to be equal to or less than 1 μ l, to be equal to or less than 0.5 μ l, to be equal to or less than 0.1 μ l or to be equal to or less than 0.01 μ l.Yet the present invention is not limited to these embodiments.In some embodiments, can use more blood, and in some embodiments, can use more blood and obtain enough serum to implement method of the present invention.

5.2RNA preparation

In one aspect of the invention, in order to measure the RNA product of biomarker of the present invention, from individual isolation of RNA.As described herein from tissue sample isolation of RNA.Sample can be from single patient or can be from a plurality of patients' merging.

In one aspect of the method, as described herein from osteoarthritis patient's blood sample direct isolation of RNA.Sample can be from single patient or can be from a plurality of patients' merging.

From tissue sample, extract total RNA according to method well-known in the art.In one embodiment, according to following method purifying RNA from tissue.After shifting out interested tissue from individuality or patient, this is organized in quick freezing in the liquid nitrogen, to prevent the degraded of RNA.In case what add certain volume organizes guanidinesalt solution, just tissue sample is come tissue abrasion's sample with twice or three times 10 pulse per second (PPS)s in tissue grinder (tissuemizer).For guanidinesalt solution (1L) is organized in preparation, the 590.8g guanidinium isothiocyanate is dissolved in the H that about 400ml handles through DEPC ₂O.The 2MTris-Cl that adds 25ml, the Na of pH 7.5 (final concentration 0.05M) and 20ml ₂EDTA (final concentration 0.01M) stirs this solution and spends the night, and volume is transferred to 950ml, and adds the 2-ME of 50ml.

With the tissue sample of homogenization under 12 ℃ of conditions centrifugal 10 minutes with 12,000 * g.Under the condition that has 0.1 volume, 20% sarcosyl, hatched resulting supernatant liquor 2 minutes at 65 ℃, form layers on the 5.7M of 9ml CsCl solution (0.1g CsCl/ml), and under 22 ℃, separate with centrifugal the spending the night of 113,000 * g.Careful remove supernatant liquor after, will manage reversing and emptying.The bottom (containing the RNA precipitation) of this pipe is placed the plastics tubing of 50ml and exists 3ml to organize resuspended damping fluid (5mM EDTA, 0.5% (v/v) sarcosyl, 5% (v/v) 2-ME) under the condition in 4 ℃ of overnight incubation (or longer time) thus make the RNA precipitation resuspended fully.According to priority, phenol/chloroform/primary isoamyl alcohol with 25: 24: 1, follow the resulting RNA solution of chloroform/primary isoamyl alcohol extracting with 24: 1, adding the sodium-acetate (pH 5.2) of 3M and 100% ethanol of 2.5 volumes precipitates, and be resuspended in (Chirgwin et al. in the DEPC water, 1979, Biochemistry, 18:5294).

As an alternative, according to following one step scheme isolation of RNA from tissue.Prepare interested tissue by homogenization in glass teflon homogenizer, wherein the tissue of every 100mg adds the denaturing soln (pH 7.0 for 4M thiosulfuric acid guanidine, 25mM Trisodium Citrate, 0.1M 2-ME, the acid of 0.5% (w/v) N-lauryl creatine) of 1ml.After homogenate is transferred to the 5ml polypropylene tube, sequentially add the 2M sodium-acetate (pH 4) of 0.1ml, chloroform/primary isoamyl alcohol of 49: 1 of the water of 1ml-saturated phenol and 0.2ml.After adding each composition, mix this sample, and after having added all the components, hatched 15 minutes at 0-4 ℃.This sample was separated with 10,000 * g under 4 ℃ in centrifugal 20 minutes, and 100% Virahol that adds 1ml precipitates, and hatches 30 minutes and precipitates in centrifugal 10 minutes with 10,000 * g under 4 ℃ at-20 ℃.The RNA that obtains precipitation is dissolved in the denaturing soln of 0.3ml, transfers in the micro-centrifuge tube ,-20 ℃ of 100% isopropanol precipitatings that add 0.3ml down 30 minutes, and under 4 ℃ with 10,000 * g centrifugal 10 minutes.In 70% ethanol, clean the RNA precipitation, drying, and be resuspended in 100-200 μ l in the water that DEPC handles or in the 0.5%SDS that DEPC handles (Chomczynski and Sacchi, 1987, Anal.Biochem., 162:156).

Preferably, under the liquid nitrogen condition, described tissue sample is processed into fine-powder and application

Reagent (GIBCO/BRL) extracts total RNA.

As an alternative, isolation of RNA from blood.In one embodiment, according to the following steps isolation of RNA.With 3 parts of lysis buffers the ratio of 1 part of blood is added lysis buffer (lysis buffer (1L) 0.6g EDTA in blood sample; 1.0g KHCO ₂, 8.2g NH ₄Cl transfers to 7.4 with NaOH with pH).Biased sample also placed 5-10 minute on ice, till transparent.Under 4 ℃,, and draw supernatant liquor with the centrifugal lysate sample of 1000rpm 10 minutes.Precipitation is resuspended in the lysis buffer of 5ml, and under 4 ℃ with 1000rmp centrifugal again 10 minutes.With about 6ml's Ratio to the original blood sample of 10ml is used

(GIBCO/BRL) with sedimentary cell homogenization and abundant vortex oscillation.Sample was placed room temperature 5 minutes.With every 1ml

Use the amount of 1.2ml chloroform and extract RNA.Under 4 ℃ with the centrifugal sample of 12,000 * g 5 minutes and collect the upper strata.With every 1ml

Ratio to 0.5ml adds Virahol to the upper strata.Sample is placed-20 ℃ spend the night or place-20 ℃ one hour.According to the known method precipitated rna, RNA is precipitated air-dry, and precipitation is resuspended in the ddH that handled through DEPC ₂O.Can also be in 75% ethanol with the RNA sample retention, this sample at room temperature is stable for transportation.

As an alternative, use as mentioned above

Reagent (GIBCO/BRL) isolation of RNA from synovia.

By the absorbancy at 260/280nm place and agarose gel electrophoresis then then the inspection under UV-light assess purity and the integrity of RNA.

5.3 biomarker of the present invention

In one embodiment, the invention provides the combination of biomarker and biomarker, measuring of the expression level of one or more products of wherein said biomarker is the index of bladder cancer.In another embodiment, the invention provides the combination of biomarker and biomarker, wherein can use the measuring of expression level of one or more products of described biomarker and diagnose individuality whether to have bladder cancer and/or commitment bladder cancer.

Table 1 provides the tabulation about the gene title of biomarker of the present invention and genes involved ID (the chain ID of previous locus), wherein can use measuring of described biomarker expression level either alone or in combination and diagnose individual for having bladder cancer; Or do not have a bladder cancer.As skilled in the art to understand, all described rna transcription things and all described proteinic sequences of can applying gene ID determining corresponding listed biomarker.

Table 2 provides the biomarker that is disclosed in PCT application No.PCT/US04/020836, its people that is can be with disclosed one or more biomarkers of table 1 combined and that come diagnosing bladder cancer or differentiation to have bladder cancer or do not have bladder cancer according to the instruction of this paper.

Table 3 discloses the reference registration number of the protein of the reference registration number of RNA product of corresponding described biomarker and correspondence table 1 listed biomarker particularly.

Table 4 provides about the gene title of biomarker of the present invention and the tabulation of genes involved ID, wherein can use measuring of described biomarker expression level either alone or in combination and diagnose individual for having the commitment bladder cancer; Or do not have a bladder cancer.As skilled in the art to understand, all described rna transcription things and all described proteinic sequences of can applying gene ID determining corresponding listed biomarker.

Table 5 provides the biomarker that is disclosed in PCT application No.PCT/US04/020836, its people that is can be with disclosed one or more biomarkers of table 4 combined and that diagnose commitment bladder cancer or differentiation to have the commitment bladder cancer or do not have bladder cancer according to the instruction of this paper.

Table 6 discloses the reference registration number of the protein of the reference registration number of RNA product of corresponding described biomarker and correspondence table 4 listed biomarkers particularly.

Table 7 discloses particularly about the gene title of biomarker of the present invention and the tabulation of genes involved ID, and described gene can be distinguished bladder cancer and non-bladder cancer.

Table 10 discloses particularly about the gene title of the biomarker of identifying in the table of selecting 1 and the tabulation of genes involved ID, and described gene can be distinguished bladder cancer and non-bladder cancer.

Table 11 provides about the gene title of the biomarker of selecting of the present invention and the tabulation of genes involved ID (the chain ID of previous locus), wherein can use measuring of described biomarker expression level either alone or in combination and diagnose individual for having bladder cancer; Or do not have a bladder cancer.As skilled in the art to understand, all described rna transcription things and all described proteinic sequences of can applying gene ID determining corresponding listed biomarker.

Table 12 discloses the reference registration number of the protein of the reference registration number of RNA product of corresponding described biomarker and correspondence table 11 listed biomarkers particularly.

The present invention is contained thus for above-mentioned each purpose, and those methods that application well known to a person skilled in the art and this paper summarizes are measured the expression of these biomarkers and biomarker combination.

5.4 biomarker combination

In one embodiment, the combination of biomarker of the present invention comprise the listed biomarker of table 1 any number, mostly be 2,3,4,5,6,7,8,10,20,30,40,50,100,125,150,175,200 or all arbitrary combination most.In another embodiment of the present invention, what the combination of biomarker of the present invention comprised arbitrary or arbitrary number mostly is 1 most, 2,3,4,5,6,7,8,10,20,30,40,50,100,125,150,175,200 or the listed biomarker of all tables 1 and arbitrary or arbitrary number mostly be 1 most, 2,3,4,5,6,7,8,10,20,30,40,50,100,250,500,750,1000,1500,2000,2500,3000,3500, any combination that 4000 or the listed biomarker of all tables 2 are combined, the expression of measuring its product can be used to diagnose individuality whether to have bladder cancer or not have bladder cancer.In another embodiment, the combination of biomarker of the present invention comprises any combination that mostly is 2,3,4,5,6,7,8,10,20,30,40,50,100,150,200,250,300,350,400,450,500,1000,1250,1500,1750,2000 or listed RNA of all tables 3 and/or protein most of arbitrary number.In another embodiment, the combination of biomarker of the present invention comprises any combination that mostly is 2,3,4,5,6,7,8,10,20,30,40,50,100,125,150,175,200,250,300,350,400,450,500 or the listed biomarker of all tables 4 most of arbitrary number.In another embodiment of the present invention, what the combination of biomarker of the present invention comprised arbitrary or arbitrary number mostly is 1 most, 2,3,4,5,6,7,8,10,20,30,40,50,100,125,150,175,200,250,300,350,400,450,500 or the listed biomarker of all tables 4 and arbitrary or arbitrary number mostly be 1 most, 2,3,4,5,6,7,8,10,20,30,40,50,100,150,200,250,300,350,400,450,500,1000,1250,1500,1750,2000, any combination that 2250 or the listed biomarker of all tables 5 are combined, the expression of measuring its product can be used to diagnose individuality whether to have early stage bladder cancer or not have bladder cancer.In another embodiment, the combination of biomarker of the present invention comprises any combination that mostly is 2,3,4,5,6,7,8,10,20,30,40,50,100,150,200,250,300,350,400,450,500,1000,1250,1500,1750,2000,2250,2500,2750,3000,3250 or listed RNA of all tables 6 and/or protein most of meaning number.In another embodiment of the present invention, the combination of biomarker of the present invention comprises any combination that mostly is 1,2,3,4,5,6,7,8,10,20,30,40,50,75,100,125,150,175,200 or the listed biomarker of all tables 7 most of arbitrary or arbitrary number.In another embodiment, the combination of biomarker of the present invention comprises any combination that mostly is 2,3,4,5,6,7,8,9 or the listed biomarker of all tables 10 most of arbitrary number.

For example, at Feller, Intro to Probability Theory, Third Edition, volume 1,1968, described the possible combined number of the subclass m of n gene among the ed.J.Wiley, and it uses following general formula:

m！/(n)！(m-n)！

In one embodiment, wherein n be 2 and m be 19, just have:

$\frac{19!}{2!(19-2)!}=\frac{19\×18\×17\×16\×15\×14\×13\×12\×11\×10\×9\×8\×7\×6\×5\×4\×3\×2\×1}{(2\×1)(19\×18\×17\×16\×15\×14\×13\×12\×11\×10\×9\×8\×7\×6\×5\×4\×3\×2\×1)}$

$=\frac{{1.21610}^{17}}{{7.1110}^{14}}=171$

Plant specific dual-gene combination.Can use independently each combination in these dual-gene combinations genetic expression measure determine whether the patient has bladder cancer.In another embodiment, wherein m be 19 and n be 3, just have 19! / 3! (19-3)! Individual specific three assortments of genes.Can be independently with each model that whether has bladder cancer as definite patient in these specific three assortments of genes.

5.5 select concrete available biomarker combination

Although the combination of the biomarker in any table of table 1-7 of the present invention and 10-13 can be respectively applied for diagnosing bladder cancer and/or early stage bladder cancer, the present invention also provides the means of selecting and assessing the biomarker combination, and described biomarker combination is from any specific table or the table pack that can be used for diagnosing bladder cancer.

In order to identify the combination of available biomarker, applied mathematical model is set up sorter (classifier), its data that can use each biomarker expression level in the reflection biomarker combination are distinguished bladder cancer and/or early stage bladder cancer and non-bladder cancer, and wherein said each biomarker is selected from those biomarkers of evaluation in table 1-7 and 10-13 one or more.The differentiation bladder cancer of assessment subsequently or evaluation sorter and/or the ability of early stage bladder cancer and non-bladder cancer, thus select energy excellent diagnostics individuality to have those sorters (and those biomarkers thus make up) of bladder cancer and/or early stage bladder cancer.

Set up sorter by using to represent the data of the expression level of the two or more selected biomarker product of each individuality in the T-group and be applied to mathematical model.Have the effectiveness of expectation in order to ensure the sorter that obtains, the phenotypic characteristic of T-group is very important.For example, in one embodiment, the T-group of application comprises two phenotype subgroups: known individuality and the known individuality that does not have bladder cancer with bladder cancer, and described biomarker is selected from table 1 and/or table 2 or table 7 or table 10 or table 11 and/or table 12.In another embodiment, the T-group of application comprises two phenotype subgroups: known individuality and the known individuality that does not have bladder cancer with commitment bladder cancer, and described biomarker is selected from table 3 and/or table 4.

The data that are input to described mathematical model can be to represent any data of biomarker product expression level.In one embodiment, the data about each biomarker that are input to described mathematical model are to use the data well known to a person skilled in the art that any method of being used for measuring gene expression dose obtains, and described measurement gene expression dose comprises the RNA product of measuring biomarker and/or the protein of measurement biomarker.In a preferred embodiment, the data that are input to mathematical model are to use the data that real-time quantitative PCR (qRT-PCR) obtains.

In order to identify especially available biomarker combination, can develop for any is used for the sorter of every kind of possible biomarker combination in table 1, table 2, table 1 and table 2 combination, table 4, table 5, table 4 and table 5 combination, table 7 or the table 10, thereby can and select those to be best suited for those evaluation sorters of diagnosing bladder cancer or early stage bladder cancer with this sorter of postevaluation.

In order to develop sorter, applied mathematical model.Can select can be used for mathematical model of the present invention from and the following: regression model, logistic regression model (logistic regression model), neural network, Clustering Model, principle component analysis, nearest neighbo(u)r classification is analyzed, linear discriminant analysis, quadratic discriminatory analysis, SVMs (Support Vector Machine), decision tree, genetic algorithm, use the classifier optimization method (classifier optimization using bagging) of bag model, use the classifier optimization method of strengthening (classifier optimization using boosting), use the classifier optimization method (classifier optimization using the randomsubspace method) of stochastic subspace method, projection pursuit method (a projection pursuit) and weighting ballot method (wighted voting).

Subsequently, according to the description evaluation of the 5th joint and/or the sorter that sorts and generate, thereby evaluation suffers from bladder cancer for the diagnosis individuality and/or early stage bladder cancer has enough susceptibility and/or specific those sorters.In order to estimate described sorter, can the inspection-classification device to determine that sorter is according to each individual ability in the correct discriminating described herein T-group.Can also differentiate that correctly the ability of one or more individualities of " estimating colony " assesses or estimate the sorter that is generated by determining sorter.It is similar estimating the T-group of describing in colony and following the 5.7th joint, but estimates colony by not using one or more individualities of generating sorter to form.Similarly, estimating colony comprises and is diagnosed as the individuality (the second phenotype subgroup) that has the individuality of bladder cancer (the first phenotype subgroup) for example and do not have bladder cancer.In one embodiment, estimate the member that colony comprises described T-group, add one or more members that are not used for described T-group.In some embodiments, be less than or equal to 5% in the described T-group, be less than or equal to 10%, be less than or equal to 20%, be less than or equal to 30%, be less than or equal to 50% or to be less than or equal to 90% member and the described colony that estimates be common.

As skilled in the art to understand, this allows the prediction sorter correctly to characterize the ability of the individuality of phenotypic characteristic the unknown.

Can use the sorter diagnosis that obtains individuality unknown or check to determine whether described check individuality has bladder cancer and/or commitment bladder cancer.In one embodiment, can use two or more sorters (" set of classifiers ") determines checking individual diagnosis.For example, in some embodiments, select the sorter of ordering preceding 10, the sorter of ordering preceding 20, the sorter of ordering preceding 100.In some embodiments, select the arbitrary classification device of ordering preceding 1 to 500.In some cases, application class device group is diagnosed, and in one embodiment, for the individual diagnosis of described check, each sorter is thrown a ticket, thereby the diagnosis of described checked object is confirmed as the result of classifiers combination.In the other embodiment, each sorter is used different weighting patterns.For example, weighting pattern can comprise the weighting based on some factors (such as the size of the coefficient of the original score of sorter, logarithm ratio ratio (logs odd ratio) (" decilog (logit) "), each sorter, their some combinations etc.).

In one embodiment, directly use sorter or set of classifiers to carry out aforesaid diagnosis.Yet the combination of having identified in another embodiment, can be independent of the sorter of firm described gene to be used.For example, can monitoring the gene expression profile that is produced by 10 genes, to assess check individual, the gene expression profile of wherein that described check is individual gene expression profile and described 10 genes of bladder cancer individuality and do not have the gene expression profile of 10 genes described in the bladder cancer individuality to compare.

5.6 select diagnosing bladder cancer and commitment bladder cancer to be particularly useful to mathematical model input data The biomarker combination

For example, to can be used for diagnosing individuality to suffer from bladder cancer or do not have those biomarkers of bladder cancer in order to identify, the individuality that the data of the expression level of one or more mRNA products of corresponding biomarker is used for T-group, it is individual and do not have the second phenotype subgroup individuality of bladder cancer to have the first phenotype subgroup of bladder cancer, and described biomarker is the biomarker in table 1 and/or table 2 and/or table 7 and/or table 10 and/or the table 11.For described T-group being characterized by the purpose of specified phenotype subgroup, can use any method of diagnosing bladder cancer.In a preferred embodiment, concerning described T-group, utilize cytoscopy to determine to diagnosis of bladder cancer.

In another embodiment, to can be used for diagnosing individuality to suffer from the commitment bladder cancer or do not have those biomarkers of bladder cancer in order to identify, the data of the expression level of one or more mRNA products of individual biomarker comprise the data that have the individual of commitment bladder cancer and do not have the individuality of bladder cancer in the reflection T-group, and described biomarker is the biomarker in table 4 and/or the table 5.

In another embodiment, the individuality that the data of expression level of one or more proteins of reflection biomarker is used for T-group, it is individual and do not have the second phenotype subgroup individuality of bladder cancer to have the first phenotype subgroup of bladder cancer, and described biomarker is the biomarker in table 1 and/or table 2 and/or table 7 and/or the table 10.For described T-group being characterized by the purpose of specified phenotype subgroup, can use any method of diagnosing bladder cancer.

In another embodiment, the individuality that the data of expression level of one or more proteins of reflection biomarker is used for T-group, it is individual and do not have the second phenotype subgroup individuality of bladder cancer to have the first phenotype subgroup of bladder cancer, and described biomarker is the biomarker in table 1 and/or table 2 and/or table 7 and/or table 10 and/or the table 11.For described T-group being characterized by the purpose of specified phenotype subgroup, can use any method of diagnosing bladder cancer.

5.7 T-group

In some embodiments, reference or T-group comprise 10-30 object.In another embodiment, described T-group comprises 30-50 object.Still in other embodiments, described reference group comprises two or more colonies, and wherein each colony comprises 50 to 100,100 to 500,500 to 1000 or surpass 1000 objects.

In some embodiments, the member of each phenotype subgroup (being bladder cancer and non-bladder cancer member) of preferred T-group so that each phenotype subgroup of T-group have about at least 1,2,3,4,5,6,1 or a plurality of, 2 or a plurality of, 3 or a plurality of, 4 or a plurality of, 5 or a plurality of, 6 or similar distribution more a plurality of, 1-1000 other phenotype.For example, age, sex, heritable variation information (existence, the shortage of for example, gene SNP sudden change, restriction fragment length polymorphism, microsatellite marker, restriction fragment length polymorphism and short series connection repeated characteristic), treatment plan; Coexistence disease (co-morbidities); Other index of the concentration of metabolite, hematochemistry level and/or health (health) and/or healthy (wellness).

5.8 mathematical model

5.8.1 regression model

In some embodiments, will be used for regression model, this base of a fruit regression model of preferred logic about the expression data of each biomarker combination to be tested.Described regression model will determine that about every kind of equation that may make up of institute's test organisms mark each equation provides and multiply by the coefficient of reflection by the data of the expression level of each individual biomarker of described model representative.

Usually, interested multiple regression equation can be written as

Y＝α+β ₁X ₁+β ₂X ₂+…+β _kX _k+ε

Wherein, dependent variable Y represent to exist (when Y on the occasion of the time) or lack (when Y is negative value) first phenotypic characteristic (for example, having mild osteoarthritis).This model shows that dependent variable Y depends on explanatory variable k (for k selection gene from the object of the first and second phenotype subgroups in the T-group, observed value is illustrated in the level of gene product described in the tissue of interest), add error term, this error term comprises the various not specially appointed omission factors.In above-mentioned model, keeping under the constant condition of other explanatory variable parameter beta ₁Represent the first explanatory variable X ₁Influence to dependent variable Y.Similarly, β ₂Provided the explanation variable X ₂To the influence of Y, keep all the other explanatory variables constant.

The logistic regression model is the nonlinear transformation of linear regression.The logistic regression model is called as " decilog (logit) " model and can be represented as

Ln[p/ (1-p)]=alpha+beta ₁X ₁+ β ₂X ₂+ ... + β _kX _k+ ε or

$[p/(1-p)]={\exp }^{\mathrm{\&alpha;}}{\exp }^{{\mathrm{\&beta;}}_1{\mathrm{<figure-callout\; id="1"\; label="X"\; filenames="A2006800240550274H1.png,A2006800240550275H1.png"\; state="\{\{state\}\}">X</figure-callout>}}_1}{\exp }^{{\mathrm{\&beta;}}_2{\mathrm{<figure-callout\; id="2"\; label="X"\; filenames="A2006800240550274H1.png,A2006800240550277H1.png"\; state="\{\{state\}\}">X</figure-callout>}}_2}\&times;\&CenterDot;\&CenterDot;\&CenterDot;\&times;{\exp }^{{\mathrm{\&beta;}}_k{X}_k}{\exp }^{\mathrm{\&epsiv;}}$

Wherein, ln is a natural logarithm, log ^Exp, exp=2.71828... wherein,

P is the probability that incident Y takes place, p (Y=1),

P/ (1-p) is " odds ratio ",

Ln[p/ (1-p)] be the logarithm odds ratio, perhaps " decilog ", and

Other all components of this model is identical with above-mentioned general regression equation.One skilled in the art will appreciate that and α can be become identical constant with the ε item.In fact, in some preferred embodiments, represent α and ε with single item." logistic " distributes is S shape distribution function.Decilog distribution limitation estimated probability (p) is between the 0-1.

In some embodiments of the present invention, by maximum likelihood estimation (MLE) match logistic regression model.In other words, determine coefficient (for example, α, β by maximum likelihood method ₁, β ₂...).Likelihood is conditionality probability (for example, the probability of Y when P (Y|X), given X).Likelihood function (L) is measured the specific dependent variable value set (Y of observation ₁, Y ₂..., Y _n) at the concentrated probability that takes place of sample data.It is represented as the probability of dependent variable product:

L＝Prob(Y ₁*Y ₂***Y _n)

Likelihood function is got over high-order, and the probability of observation Y in sample is big more.MLE relates to and finds make the logarithm of likelihood function as far as possible equally big (LL＜0) or make the logarithm of likelihood function as far as possible little-2 times of (coefficient 2LL) (α, β ₁, β ₂...).In MLE, to parameter alpha, β ₁, β ₂... carry out some initial estimations.Calculate the likelihood of the data of determining these parameter estirmation subsequently.The likelihood that recomputates described data improves the estimation of parameter.Repeat this process, till the not too many variation of parameter estirmation (for example, probability changes less than 0.01 or 0.001).At Hastie, The Elementsof Statistical Learning, Springer, New York, 2001, pp.95-100 can find the example of logistic recurrence and match logistic regression model, and its integral body is incorporated this paper into by reference.

5.8.2 neural network

In another embodiment, can utilize measurement to come neural network training to the expression of each biomarker of the present invention.Neural network is that a kind of two rank return or disaggregated model.Neural network has layered structure, and it comprises input block (input units) (and bias (the bias)) layer, and this layer links to each other with the output unit layer by weight layer (layer of weights).For recurrence, the output unit layer generally includes just what a output unit.Yet neural network can be handled a lot of quantitative responses with seamless way.Similarly, can use neural network to distinguish the biomarker that surpasses two kind of groups to identify.In a specific embodiment, can use expression data neural network training from the product of the described biomarker of table 1 (being included in those biomarkers of pointing out in the table 3), thereby identifying to compare does not have bladder cancer, and bladder cancer had specific those biomarkers combinations.As a result of, can use tested neural network and directly identify the biomarker combination that can be used as the phasic specificity biomarker.In some embodiments, the reverse transmittance nerve network of using in the EasyNN-Plus4.0g version software package (NeuralPlanner Software Inc.) that comprises the single hiding layer of ten neurones (ten hidden units) (is seen, Abdi for example, 1994, " A neural network primer ", J.Biol.System.2,247-283).

At Duda et al., 2001, Pattern Classification, Second Edition, JohnWiley ﹠amp; Sons, Inc., New York; With Hastie et al., 2001, The Elements ofStatistical Learning, Springer-Verlag has described neural network among the New York, and its integral body is incorporated this paper into reference.

5.8.3 other mathematical model

Above-mentioned pattern classification and statistical technique only are the examples of some type models, it can be used to make up the model at bladder cancer, for example at Duda and Hart, and Pattern Classification andScene Analysis, 1973, John Wiley ﹠amp; Sons, Inc., the cluster of describing in the 211-256 page or leaf of New York (its integral body is incorporated this paper into by reference); Principle component analysis (see for example Jolliffe, 1986, Principal Component Analysis, Springer, New York, it incorporates this paper into by reference); Nearest neighbo(u)r classification analysis (is seen for example Duda, Pattern Classification, Second Edition, 2001, John Wiley ﹠amp; Sons, Inc; And Hastie, 2001, The Elements of StatisticalLearning, Springer, New York); Linear discriminant analysis (is seen for example Duda, PatternClassification, Second Edition, 2001, John Wiley ﹠amp; Sons, Inc; And Hastie, 2001, The Elements of Statistical Learning, Springer, New York; Venables ﹠amp; Ripley, 1997, Modern Applied Statistics with s-plus, Springer, New York); SVMs (is seen for example Cristianini and Shawe-Taylor, 2000, An Introduction toSupport Vector Machines, Cambridge University Press, Cambridge, Boseret al, 1992, " A training algorithm for optimal margin classifiers, inProceedings of the 5 ^ThAnnual ACM Workshop on Computational LearningTheory, ACM Press, Pittsburgh, PA, pp.142-152; Vapnik, 1998, StatisticalLearning Theory, Wiley, New York, all described documents are incorporated this paper into by reference).

5.9 select biomarker to make up the selection sort device

Preferred all biomarkers of selecting are used in subsequent portion.Therefore, the data of using the product level of representing each biomarker are assessed all possible biomarker combination.In some embodiments, can not select all possible combination, therefore at first select the subclass of biomarker and in the next combination of applying biological mark subclass.As is understood, based on the desired number of selected biomarker, choice criteria will depend on and obtain represent the available source of biomarker product horizontal data and/or calculate with the available computers of the selected biomarker of assessment all or the sorter that partly may make up and originate.In some embodiments, the desired number of the biomarker of the selection that can assess is 4,000; 3,000; 2,000; 1,000; 900; 800; 700; 600; 500; 400; 300; 200; 190; 180; 170; 160; 150; 140; 130; 120; 110; 100; 90; 80; 70; 60; 50; 40; 30; 20; 10.

In one embodiment of the invention, the various of biomarker may make up in the evaluation form 1.In another embodiment of the present invention, 2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. the various of biomarker of evaluation form 1 may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 1 all biomarkers may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 1 part biological mark may make up.In another embodiment of the present invention, table 1 part biological of being assessed is labeled as the biomarker of 20,30,40,50,60,70,80,90,100,125,150,175,200,225 or 250 tables 1.Still in another embodiment of the present invention, sort according to the part biological mark of individual p value to selected table 1, the single differentiation of wherein said each biomarker of p value representation has the member of bladder cancer and does not have bladder cancer member's ability, select subsequently through the ordering biomarker preceding 60,50,40,30,20 or 10 individuality also utilizes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of selected all biomarkers to create sorter, and the assessment sorter is distinguished the ability that has the member of bladder cancer and do not have the bladder cancer member.Still in another embodiment, the subclass of option table 1 biomarker, their p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, the differential expression level of between two or more feature subgroups, the showing subclass of coming option table 1 biomarker according to described biomarker product.In the time of should noting the difference multiple variation in measuring blood, the difference that multiple changes may be very little, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, using one or more choice criteria and ordering subsequently allows to select preceding 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker of ordering artifact mark to form sorter.In another embodiment of the present invention, be condition in the combination of being assessed, to comprise at least one biomarker from table 1, the various of evaluation form 1 and table 2 biomarker may make up.In another embodiment of the present invention, 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. the various of biomarker of 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. biomarker combination table 2 of evaluation form 1 may make up.In another embodiment of the present invention, the exhaustive search of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarker combinations of all biomarkers of evaluation form 1 and table 2 combination.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the part biological mark of evaluation form 1 and table 2 may make up.In another embodiment of the present invention, the table of being assessed 1 and the part biological of table 2 are labeled as the combination of a biomarker such as 1,2,3,4,5,6,7,8,9,10,20,30,40,50,75,100,150,200,300 of 20,30,40,50,60,70,80,90,100,125,150,175,200,225 or 250 biomarkers of table 1 and table 2.Still in another embodiment of the present invention, the part biological mark of selected table 1 and table 2 sorts according to the p value of individuality, and the single differentiation of wherein said each biomarker of p value representation has the member of bladder cancer and do not have bladder cancer member's ability.Still in another embodiment, the subclass of option table 1 and table 2 biomarker, its p value is less than 0.5; Less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, according to the horizontal option table of showing by the biomarker product 1 of differential expression and the subclass of table 2 biomarker.It should be noted when the difference multiple changes in measuring blood, the difference that multiple changes may be very little, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, use one or more choice criteria and ordering subsequently and allow to select preceding 2.5% of the biomarker that sorts, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the biomarker utilize all selections create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.

In another embodiment of the present invention, the various of biomarker may make up in the evaluation form 4.In another embodiment of the present invention, 2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,150,200,250,300,350,400,450,500 etc. the various of biomarker of evaluation form 4 may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 4 all biomarkers may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 4 part biological marks may make up.In another embodiment of the present invention, table 4 part biological of being assessed is labeled as the biomarker of 20,30,40,50,60,70,80,90,100,125,150,175,200,225,250,300,350,400,450 or 500 tables 4.Still in another embodiment of the present invention, sort according to the p value of individuality part biological mark to selected table 4, the single differentiation of wherein said each biomarker of p value representation has the member of superficial bladder cancer and does not have bladder cancer member's ability, select subsequently through the ordering biomarker preceding 60,50,40,30,20 or 10 individuality also utilizes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of selected all biomarkers to create sorter, and the assessment sorter is distinguished the ability that has the member of superficial bladder cancer and do not have the bladder cancer member.Still in another embodiment of the present invention, the part biological mark of option table 4, its p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, according to the differential expression level of being showed by described biomarker product, the subclass of option table 4 biomarkers is formed for the combination of input category device.In the time of should noticing that the difference multiple changes in measuring blood, multiple changes difference may be very little, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore, in some embodiments,,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker at first according to the p value that produces by biomarkcr data.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, use one or more choice criteria and ordering subsequently and allow to select preceding 2.5% of the biomarker that sorts, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the biomarker utilize all selections create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.In another embodiment of the present invention, be condition in the combination of assessment, to comprise at least one biomarker from table 4, the various of evaluation form 4 and table 5 biomarker may make up.In another embodiment of the present invention, individual combined various may the combinations of biomarker such as 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 in 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. biomarkers and the table 5 in the evaluation form 4.In another embodiment of the present invention, the exhaustive search of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarker combinations of all biomarkers of evaluation form 4 and table 5 combination.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the part biological mark of evaluation form 4 and table 5 may make up.In another embodiment of the present invention, the table of being assessed 4 and the part biological of table 5 are labeled as the combination of a biomarker such as 1,2,3,4,5,6,7,8,9,10,20,30,40,50,75,100,150,200,300,500,1000,1250,1500,1750,2000,2250 of 20,30,40,50,60,70,80,90,100,125,150,175,200,225 or 250,300,350,400,450,500 biomarkers of table 4 and table 5.Still in another embodiment of the present invention, sort according to the p value of individuality part biological mark to selected table 4, the single differentiation of wherein said each biomarker of p value representation has the member of superficial bladder cancer and does not have bladder cancer member's ability, select subsequently through the ordering biomarker preceding 60,50,40,30,20 or 10 individuality also utilizes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of selected all biomarkers to create sorter, and the assessment sorter is distinguished the ability that has the member of superficial bladder cancer and do not have the bladder cancer member.Still in another embodiment of the present invention, the biomarker of option table 4 and table 5 part, their p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, according to the biomarker subclass of horizontal option table of showing by described biomarker product 4 of differential expression and/or table 5 to be formed for the combination of input category device.In the time of should noticing that the difference multiple changes in measuring blood, described multiple changes difference may be very little, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, using one or more choice criteria and ordering subsequently allows to select through preceding 2.5% of ordering biomarker, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers utilizing selected all biomarkers create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.

In another embodiment of the present invention, the various of biomarker may make up in the evaluation form 7.In another embodiment of the present invention, 2,3,4,5,10,20,30,40,50,60,70,80,90,100,125,150,175,200 etc. the various of biomarker of evaluation form 7 may make up.In another embodiment of the present invention, biomarker according to p value (as described in Table 1) his-and-hers watches 7 of individuality sorts, the single differentiation of wherein said each biomarker of p value representation has the member of bladder cancer and does not have bladder cancer member's ability, the institute that assesses subsequently through preceding 60,50,40,30,20 or 10 individualities of the biomarker of ordering might make up, thereby selects to distinguish the member with bladder cancer and do not have those combinations of bladder cancer member.Still in another embodiment, utilize the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of all biomarkers of table 7 to create sorter, described sorter is distinguished bladder cancer and non-bladder cancer.Still in another embodiment, sorter is created in 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 combinations of maximum 2,3,4,5,10,20,30,40,50,60,70,80,90,100,125,150,175 or 200 biomarkers of application table 7, assesses the ability that described sorter is distinguished bladder cancer and non-bladder cancer subsequently.Still in another embodiment, sorter is created in 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 combinations of application table 7 preceding 200,175,150,125,100,90,80,70,60,50,40,30,20 or 10 kind of biomarker (based on the p value), assesses the ability that described sorter is distinguished bladder cancer and non-bladder cancer subsequently.Still in another embodiment of the present invention, the part biological mark of option table 7, their p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, the differential expression level of showing according to described biomarker product comes option table 7 biomarker subclass to be formed for the combination of input category device.In the time of should noticing that the difference multiple changes in measuring blood, it may be very little that described multiple changes difference, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, using one or more choice criteria and ordering subsequently allows to select through preceding 2.5% of ordering biomarker, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers utilizing selected all biomarkers create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.

Still in another embodiment, utilize all of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of all biomarkers of table 10 to make up and create sorter, this sorter can be distinguished bladder cancer and non-bladder cancer.

In another embodiment of the present invention, 2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. the various of biomarker of evaluation form 11 biomarkers may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 11 all biomarkers may make up.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of evaluation form 11 part biological marks may make up.In another embodiment of the present invention, the part biological mark of the table of being assessed 11 is 20,30,40,50,60,70,80,90,100,125,150,175,200,225 or 250 biomarkers of table 11.Still in another embodiment of the present invention, sort according to the p value of individuality part biological mark to selected table 11, the single differentiation of wherein said each biomarker of p value representation has the member of bladder cancer and does not have bladder cancer member's ability, select subsequently through the ordering biomarker preceding 60,50,40,30,20 or 10 individuality also utilizes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of selected all biomarkers to create sorter, and the assessment sorter is distinguished the ability that has the member of bladder cancer and do not have the bladder cancer member.Still in another embodiment of the present invention, the part biological mark of option table 11, their p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, the differential expression level of showing according to described biomarker product comes the subclass of option table 11 biomarkers to be formed for the combination of input category device.In the time of should noticing that the difference multiple changes in measuring blood, it may be very little that described multiple changes difference, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, using one or more choice criteria and ordering subsequently allows to select through preceding 2.5% of ordering biomarker, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the biomarker utilize all selections create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.In another embodiment of the present invention, be condition in the combination of assessment, to comprise at least one biomarker from table 11, the various of biomarker of evaluation form 11 and table 2 combination may make up.In another embodiment of the present invention, the various of biomarker such as 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 of 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60 etc. biomarkers of evaluation form 11 and combination table 2 may make up.In another embodiment of the present invention, the exhaustive search of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarker combinations of all biomarkers of evaluation form 1 and table 2 combination.In another embodiment of the present invention, the various of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of the part biological mark of evaluation form 11 and table 2 may make up.In another embodiment of the present invention, the table of being assessed 11 and the part biological of table 2 are labeled as the combination of a biomarker such as 1,2,3,4,5,6,7,8,9,10,20,30,40,50,75,100,150,200,300 of 20,30,40,50,60,70,80,90,100,125,150,175,200,225 or 250 biomarkers of table 11 and table 2.Still in another embodiment of the present invention, sort according to the p value of individuality part biological mark to selected table 11, the single differentiation of wherein said each biomarker of p value representation has the member of bladder cancer and does not have bladder cancer member's ability, select subsequently through the ordering biomarker preceding 60,50,40,30,20 or 10 individuality also utilizes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers of selected all biomarkers to create sorter, and the assessment sorter is distinguished the ability that has the member of bladder cancer and do not have the bladder cancer member.Still in another embodiment of the present invention, the part biological mark of option table 11 and table 2, their p value are less than 0.5, less than 0.1, less than 0.05, less than 0.01, less than 0.005, less than 0.001, less than 0.0005, less than 0.0001, less than 0.00005, less than 0.00001, less than 0.000005, less than 0.000001 etc.In some embodiments, according to the biomarker subclass of horizontal option table of showing by described biomarker product 11 of differential expression and/or table 2 to be formed for the combination of input category device.In the time of should noticing that the difference multiple changes in measuring blood, described multiple changes difference may be very little, therefore in some embodiments, selection to biomarker is based on the variation of difference multiple, and wherein said multiple changes greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, greater than 1.7, greater than 1.8, greater than 1.9, greater than 2.0, greater than 2.1, greater than 2.2, greater than 2.3, greater than 2.4, greater than 2.5, greater than 2.6, greater than 2.7, greater than 2.8, greater than 2.9, greater than 3.0, greater than 3.1, greater than 3.2, greater than 3.3, greater than 3.4, greater than 3.5, greater than 4.0 etc.In some embodiments, as skilled in the art to understand, help to select biomarker based on p value and multiple variation formation combination.Therefore in some embodiments, at first according to the p value that produces by biomarkcr data,, change according to the difference multiple of determining from biomarkcr data subsequently described biomarker is selected again according to above-mentioned selection biomarker.In other embodiments, at first change and select biomarker, select again according to the p value subsequently based on the difference multiple.In some embodiments, using one or more choice criteria and ordering subsequently allows to select through preceding 2.5% of ordering biomarker, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 30%, 40%, 50% or more biomarker use, this makes various may the combination of 2 and/or 3 and/or 4 and/or 5 and/or 6 and/or 7 and/or 8 and/or 9 and/or 10 biomarkers utilizing selected all biomarkers create sorter, and assesses described sorter and distinguish the ability that has the member of bladder cancer and do not have the bladder cancer member.

5.10 assessment sorter

In case applied mathematical model has calculated one or more sorters, can assess described sorter to determine which sorter is the most effective for its intended purposes.In a preferred embodiment of the invention, assess or estimate each sorter and correctly characterize the ability that each individuality in the T-group has bladder cancer or do not have bladder cancer.For example, can use jackknife analysis (jackknife analysis) and other disclosed method that cross validation Fa-Liu one cross validation (Leave One out Cross Validation), n-overlap fork checking (n-fold crossvalidation), application standard statistical method and assess described sorter.As used herein, the process of assessing described sorter is called as " estimating (scoring) ".In some embodiments, application training colony implementation evaluation.In other embodiments, according to application described herein " evaluation colony (scoring population) " implementation evaluation.Still in other embodiments, not only use " T-group " but also use " estimating colony " and come implementation evaluation.In one embodiment, described evaluation colony also comprises the member of described T-group except that comprising one or more members that are not used for T-group.In some embodiments, have be equal to or less than 5%, be equal to or less than 10%, be equal to or less than 20%, be equal to or less than 30%, be equal to or less than 50% or the member and the described evaluation colony that are equal to or less than 90% described T-group be common.

In some embodiments, percent utilization is proofreaied and correct each sorter of prediction (percent correctpredictions) statistical appraisal." percentage corrections prediction " statistical assumption is if the p that estimates expects that so described incident will take place, otherwise do not take place more than or equal to 0.5.By distributing these probability is 0 and 1, the value of sample in the T-group is compared, thereby the sample of determining to gather how much percentage ratio of T-group is correct.

In one embodiment, being used for assessing sorter, correctly to characterize in the T-group method of each individual capability be the described sorter susceptibility (TPF of assessment, True Positive Rate (true positivefraction)) and the method for 1-specificity (TNF, true negative rate (true negative fraction)).For example, in one embodiment, use susceptor operating characteristic (Receiver OperatingCharacteristic) (" ROC ").ROC provides Several Parameters to assess the susceptibility and the specificity of the diagnostic result of described equation generation.For example, in one embodiment, use ROC area (area under curve) and assess described equation.In a preferred embodiment, the ROC area is greater than the 0.5,0.6,0.7,0.8, the 0.9th, and is preferred.On the other hand, the ideal ROC cartographic represenation of area of score 1.0 has 100% susceptibility and 100% specificity.

According to the understanding of various equivalent modifications, the two-dimensional signal that area under curve will be included in the ROC curve converts one-dimension information to.In other embodiments, directly be used to information from ROC curve two dimension aspect.For example, the ROC curve also provides relevant sorter susceptibility and specific information.In some embodiments, based on susceptibility or specificity selection sort device.This can be an important evaluating index.For example, for being ill with individual mistaken diagnosis but not individual mistaken diagnosis is safer situation just often, the diagnostic classification device with high specific (promptly the false negative of a small amount of) can be important.Therefore, in some embodiments, can be provided with about susceptibility or specific cutoff value and according to surplus variable and sorter is sorted or estimate.In some embodiments, use the ROC curve that any currently known methods that produces data produces each sorter.In some embodiments, use microarray and produce data.In some embodiments, utilize quantitative RT-PCR to produce data.

In some embodiments, sorter is the logistic regression model of weighting, it is characterized in that many categories decilog model.For example, in some embodiments, sorter is distinguished two different characteristics groups.In other embodiments, sorter is distinguished and is surpassed two different characteristics group.At Agresti, An Introduction to Categorical Data Analysis, John Wiley ﹠amp; Sons, Inc., 1996, New York has described the decilog model in the 7th and 8 chapters, comprises many categories decilog model, and the document is incorporated this paper into by reference.Table 8 has exemplified based on expression data, is used for forming the data of ROC curve, and these data are used to use the data model of decilog:

ln[p/(1-p)]＝α+β ₁X ₁+β ₂X ₂+ε

Table 8: decilog ln[p/ (1-p)]=alpha+beta ₁X ₁+ β ₂X ₂The value of+ε

Different sample in the corresponding evaluation of each row colony of table 8.Left column is represented the result about the decilog that is sampled sorter.Sort according to the sample in the decilog score value his-and-hers watches 8 of listing in the left hand row.Right-hand column is specifically understood the existence of feature or lacks that described feature is to consider in regression equation.Can application table 8 calculate the ROC curve, wherein in order to calculate the ROC curve data point, each row of considering table 8 is as the threshold cutoff value.Then, in order to assess the prediction character of sorter, can calculate the ROC area under a curve.

5.11 the product of biomarker of the present invention

As understood by a person skilled in the art, identify the combination of one or more biomarkers, described biomarker is differential expression between bladder cancer and non-bladder cancer, permission is carried out the bladder cancer diagnosis, the data of the expression level of the product that the biomarker (gene) that wherein uses reflection to be identified makes up to checking individuality.

The product of each biomarker of the present invention comprises RNA and protein.The RNA product of biomarker of the present invention is the transcription product of biomarker of the present invention and comprises hnRNA, mRNA and the colony of one or more mRNA splice variants.For putting into practice the present invention, the measurement to one or more RNA product colonies of biomarker of the present invention can be used for diagnostic purpose.More particularly, the present invention comprises the measurement to those RNA product colonies of described biomarker, and described biomarker product is differential expression between bladder cancer and/or early stage bladder cancer and non-bladder cancer.

In one embodiment of the invention, the RNA product of measured biomarker of the present invention is the colony of RNA product, comprises all splice variants of hnRNA, mRNA and described mRNA.In another embodiment, the RNA product of measured biomarker of the present invention is the colony of mRNA.In another embodiment of the present invention, the RNA product of measured biomarker of the present invention is the mRNA colony of expressing in blood.Still in another embodiment of the present invention, the RNA product of measured biomarker of the present invention is the colony of one or more splice variants of described mRNA.Still in another embodiment of the present invention, the RNA product of measured biomarker of the present invention is the colony of one or more splice variants of the mRNA that expresses in blood.In another embodiment, the RNA product of biomarker of the present invention is all mRNA corresponding to genes identified seat link (gene I) in table 1-2,4-5, arbitrary table of 7 and 10.In another embodiment of the present invention, the RNA product of measured biomarker of the present invention is the RNA product corresponding to locus link (locus link) (gene I) in arbitrary table of table 3 and 6.In another embodiment of the present invention, the RNA product of measured biomarker of the present invention is those RNA products corresponding to the locus link of expressing in blood (gene I).

Scope of the present invention also comprises the protein of biomarker of the present invention and comprises all protein product colony that is produced by biomarker of the present invention.As skilled in the art to understand, all protein colony that is produced by biomarker of the present invention comprises protein, the protein variant that is produced by the mRNA variant of montage and through the protein of posttranslational modification.In one embodiment, the protein of biomarker of the present invention be corresponding to table 1-2,4-5,7 and arbitrary table of 10-11 in all proteins of genes identified seat link (gene I).In another embodiment of the present invention, the protein of measured biomarker of the present invention is the protein corresponding to locus link (gene I) in arbitrary table of table 3 and 6.In another embodiment of the present invention, the protein of measured biomarker of the present invention is those protein corresponding to the locus link of expressing in blood (gene I).For putting into practice the present invention, the measurement to one or more protein colonies of biomarker of the present invention can be used for diagnostic purpose.More particularly, this paper has comprised the measurement of those protein colonies of described biomarker and can be used for diagnostic purpose, and the protein of described biomarker is not expressed with there being the bladder cancer interindividual variation at the individuality with bladder cancer and/or commitment bladder cancer.

In one embodiment of the invention, the protein of measured biomarker of the present invention is the colony of all protein product that produces from the translation of the RNA product of biomarker of the present invention.In another embodiment, the protein of biomarker of the present invention is those proteins of expressing in blood.Still in another embodiment of the present invention, protein arbitrary or multiple biomarker of the present invention is the arbitrary or multiple proteins product that comes from arbitrary or multiple mRNA splice variant translation.Still in another embodiment of the present invention, the protein of biomarker of the present invention is the arbitrary or multiple proteins product that comes from the arbitrary or multiple mRNA splice variant translation of expressing blood.

5.12 application combination identifies diagnosing bladder cancer and/or commitment bladder cancer

As described herein, utilization has been used mathematical model corresponding to the data of the expression level of each biomarker of institute's test organisms marker combination (for example logistic recurrence etc.) and has been created sorter.On behalf of the data-switching of institute's each biomarker expression level of test organisms marker combination, sorter applied mathematics function will become to determine the individual diagnostic result that whether has bladder cancer and/or commitment bladder.By determining the result, can directly diagnose individuality whether to have bladder cancer and/or early stage bladder cancer by the application class device about checking individual data input category device or each sorter in the set of classifiers to produce diagnosis.For example, this base of a fruit of applied logic returns creates described sorter, and described sorter adopts following form:

Y＝α+β ₁X ₁+β ₂X ₂+…+β _kX _k+ε

The X of equation wherein ₁, X ₂... X _kThe observed value of expression, the representative gene product level of the k option biomarker of the relevant described combination that is used for producing sorter in interested tissue.Therefore for the diagnostic check individuality, the observed value input and the Y value of each biomarker of relevant described equation are determined the described diagnostic result of checking individuality.

The application class device makes up evaluation individually.For example, if the sorter of having selected to use three biomarkers (for example, have the ROC area under curve and must be divided into 0.9, this shows hypersensitivity and high specific), can measure each product abundance of relevant described three biomarkers in the check individuality so also compares each abundance measurement value and one or more individuality of described three biomarkers, described individuality has the control population of bladder cancer and/or early stage bladder cancer from individuality, do not have the control population of bladder cancer from individuality, thereby determine that the individual expression pattern of checking is more similar or more similar to the contrast that does not have bladder cancer to the contrast of bladder cancer and/or early stage bladder cancer.In a preferred embodiment, for example, the RNA that the biomarker of being identified in the check individuality by measurement makes up and/or the level of protein are also imported described sorter, just can use the sorter of generation and diagnose individuality.In one embodiment, use identical method and produce expression data, be used for the mathematical model of diagnostic check individuality with described expression data generation.

5.13 identifying, application combination monitors disappearing of bladder cancer

The present invention instructs and identifies that the available biomarker combination and the sorter of relevant those combinations are used to the ability of diagnosing individuality to have the commitment bladder cancer or not having the bladder cancer purpose.It will be understood by those skilled in the art that compares with the situation that does not have bladder cancer is used for diagnosing the combination of commitment bladder cancer and sorter also to can be used for determining individually disappear whether having aspect the seriousness of their bladder cancer, for example to the reaction of treatment.For example, use one or more combinations and identify, before treatment, individuality can be accredited as and have the commitment bladder cancer.After treatment, can check individual once more to determine whether described individuality still has the commitment bladder cancer.If individuality can no longer be accredited as the treatment last stage, this itself can show that treatment is effective.In addition, treatment can cause disappearing of bladder cancer stage, and this makes diagnosis now individual for not having bladder cancer.

5.14 use the product that polynucleotide are measured biomarker of the present invention

According to measuring the means that biomarker RNA product of the present invention is expressed, can use following one or multinomial, understanding according to those skilled in the art, measure the expression of RNA in the sample of the present invention in conjunction with one or more methods: other combination of oligonucleotide, cDNA, DNA, RNA, PCR product, synthetic DNA, synthetic RNA or natural modified nucleotide, its specific hybrid is to one or more RNA products of biomarker of the present invention.In another embodiment, use other combination of the oligonucleotide of oligonucleotide, cDNA, DNA, RNA, PCR product, synthetic DNA, synthetic RNA or natural modified nucleotide, its specific hybrid is to one or more RNA products of biomarker of the present invention.In a preferred embodiment, use other combination of the modified oligonucleotide of oligonucleotide, cDNA, DNA, RNA, PCR product, synthetic DNA, synthetic RNA or natural Nucleotide, it had not only hybridized one or more RNA products to biomarker of the present invention specifically but also optionally.

5.15 measure the technology of the RNA product of biomarker of the present invention

Hybridization array

In one embodiment of the invention, can use and be used for measuring the polynucleotide conduct of RNA product of the present invention and the nucleic acid member of upholder stable bond, comprise array at upholder described in one aspect of the invention.Nucleic acid member's length range can and select to have specific nucleic acid member for the RNA product of biomarker of the present invention for 8-1000 Nucleotide length.In one embodiment, these members have specificity and/or selectivity to the RNA product of biomarker of the present invention.Still in another embodiment, these members have specificity and/or selectivity to the mRNA product of biomarker of the present invention.In a preferred embodiment, these members have specificity and/or selectivity to all variants of the mRNA product of biomarker of the present invention.Still in another preferred embodiment, these members have specificity and/or selectivity to one or more variants of the mRNA variant of biomarker of the present invention.Described nucleic acid member can be strand or double-stranded, and/or can be oligonucleotide or from cDNA amplification PCR fragment.Preferably, the about 20-30 of an oligonucleotide Nucleotide is long.The preferred 100-600 of an EST Nucleotide is long.It will be appreciated by those skilled in the art that the part expression zone that can utilize biomarker of the present invention is as the probe on the array.More specifically, be complementary to gene of the present invention and/or come from the cDNA of gene of the present invention or the oligonucleotide of EST is an available.In some embodiments of the present invention, can be specifically and/or optionally hybridize to the polynucleotide of the RNA product of biomarker of the present invention and can be used on the array of the present invention by point.For the array based on oligonucleotide, selecting the oligonucleotide that can be used as probe corresponding to gene of interest is well-known in the art.More specifically, it is very important selecting to allow the zone of hybridization target nucleic acid.Tm, GC percentage composition, the degree of secondary structure and some factors of length nucleic acid such as oligonucleotide are important factors.See for example U.S. Patent No. 6,551,784.In one embodiment, described array is made up of long can the hybridizing to the sequence of one or more products of each biomarker of the present invention of 10-1000 Nucleotide, described biomarker of the present invention is disclosed in table 1, table 2, table 4, table 5 and table 7 and/or table 10 and/or table 11, comprises specific RNA and/or protein that those are pointed out in table 3 and table 6 and/or table 12.

With hybridization array of the present invention and the target nucleic acid sample analyzed preferably from human cartilage, blood or synovia.The RNA available quantity that is used as the target nucleic acid sample that is limited in about this process.Preferably, for application of the present invention, need to obtain total RNA of at least 1 microgram.With PCR with at primer (for example, the poly T oligonucleotide) combination of mRNA subspecies, can use more a spot of RNA.

The preparation of target

The target of array of the present invention preferably comes from human blood and/or human bladder tissue.

Target nucleic acid is can the bind nucleic acid probe or nucleic acid member's complementary sequence, and described combination is the chemical bond by one or more types, by the complementary base pairing, passes through hydrogen bonded usually usually.

As used herein, " come from the nucleic acid of mRNA transcript " or " corresponding to the nucleic acid of mRNA " is meant fully with synthetic mRNA transcript or its subsequence nucleic acid as template.Therefore, the RNA that transcribes from the cDNA of mRNA reverse transcription, from described cDNA, the RNA that transcribes from the DNA of described cDNA amplification, from the DNA of described amplification etc. come from or corresponding to described mRNA transcript, and these are derived or the detection indication of corresponding product or indicate the existence and/or the abundance of primary transcript described in the sample in proportion.Therefore, suitable target nucleic acid sample include but not limited to the mRNA transcript of one or more genes, the cRNA that transcribes from the cDNA of described mRNA reverse transcription, from described cDNA, the RNA that transcribes from the DNA of one or more gene amplifications, from the DNA of amplification etc.The nucleic acid target preferred source of Ying Yonging is in human cartilage, blood or synovia herein.Preferably, described target is the nucleic acid that comes from human cartilage, blood or synovia extract.Nucleic acid can be to use method well known in the art (for example, reverse transcription or PCR) from human cartilage, blood or synovia mRNA extract synthetic strand or double-stranded DNA, RNA or DNA RNA hybrid.

In the simplest embodiment, such nucleic acid target comprise total mRNA or corresponding to mRNA (for example, nucleic acid samples cDNA), described mRNA separates self-organization or blood sample.In another embodiment, application examples such as sour guanidine-phenol-chloroform extraction method is separated total mRNA from specified sample, and (see by oligomerization dT column chromatography or by using (dT) n magnetic bead separation poly A+mRNA, for example, Sambrook et al., Molecular Cloning:A LaboratoryManual (2nd ed.), Vols.1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F.Ausubel et al., ed.GreenePublishing and Wiley-Interscience, New York (1987)).In a preferred embodiment, use

(GIBCO/BRL, Invitrogen Life Technologies Cat.No.15596) extract total RNA to reagent.Check purity and the integrity of assessing RNA under the UV-light by following at the absorbancy at 260/280nm place and agarose gel electrophoresis.

In some embodiments, the amplifying target nucleic acid sample is an ideal before hybridization, for example when using limited amount organizing.It will be understood by those of skill in the art that no matter use which kind of amplification method,, must be noted that the method for using the relative frequency of keeping or control institute's amplification of nucleic acid if quantitative result is an ideal." quantitatively " amplification method is well-known for those skilled in the art.For example, quantitative PCR comprises the control sequence of using identical primer while coamplification known quantity.This provides internal standard, and it can be used to calibrate the PCR reaction.High density arrays can comprise subsequently has specific probe to described internal standard, and described interior mark is used for the quantitative nucleic acid that is increased.At PCRProtocols, A Guide to Methods and Applications, Innis et al., Academic Press, Inc.N.Y. provides the detailed protocol of quantitative PCR in (1990).

Other suitable amplification method includes but not limited to polymerase chain reaction (PCR) (Innis, etal., PCR Protocols.A Guide to Methods and Application.Academic Press, Inc.San Diego, (1990)), ligase chain reaction (LCR) (LCR) (is seen Wu and Wallace, 1989, Genomics, 4:560; Landegren, et al., 1988, Science, 241:1077 and Barringer, et al., 1990, Gene, 89:117, transcription amplification (Kwoh, et al., 1989, Proc.Natl.Acad.Sci USA, 86:1173) and self-sustained sequence replication (Guatelli, et al., 1990, Proc.Nat.Acad.Sci.USA, 87:1874).

In an especially preferred embodiment, the primer reverse transcription target nucleic acid sample mRNA that uses reversed transcriptive enzyme and form by the sequence of oligomerization dT and coding phage t7 promotor, thus the single stranded DNA template is provided.Use archaeal dna polymerase polymerization the 2nd DNA chain.After the synthetic double chain cDNA, add t7 rna polymerase and from described cDNA template transcribe rna.Continuous several wheel the to each single cDNA template transcribed the RNA that produces amplification.The in-vitro transcription method is well-knownly (to see for those skilled in the art, for example, above-mentioned Sambrook) and this ad hoc approach at VanGelder, et al., 1990, Proc.Natl.Acad.Sci.USA has detailed description among the 87:1663-1667, it shows that in this way amplification in vitro has kept the relative frequency of various rna transcription things.In addition, Eberwine et al.Proc.Natl.Acad.Sci.USA, 89:3010-3014 provides a kind of scheme, thus it is used two-wheeled and realizes that via the amplification of in-vitro transcription initial starting material are greater than 10 ⁶Amplification doubly, thus, even in biological sample is limited situation, also allow detection of expression.

Mark target or nucleic acid probe

Can mark target or probe.

But can use the mark of any analyzing and testing in the present invention, this mark is attached to or is integrated in molecule.But the analyzing and testing mark is meant detected by analysis and quantitative any molecule, part or atom.

The detectable label that is suitable for the present invention's application comprises can pass through any composition that spectrum, photochemistry, biological chemistry, immunochemistry, electricity, optics or chemical means detect.Available mark of the present invention comprises the painted vitamin H of streptavidin conjugate, magnetic bead (for example, the Dynabeads with mark ^TM), fluorescence dye (for example, fluorescein, Texas red (texas red), rhodamine, green fluorescent protein etc.), radio-labeling (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), enzyme (for example, horseradish peroxidase, alkaline phosphatase and in ELISA other enzyme commonly used) and color development mark are such as Radioactive colloidal gold or tinted shade or plastics (for example, polystyrene, polypropylene, latex etc.) pearl.Some patents have been instructed the application of this class mark, comprise U.S. Patent No. 3,817, and 837,3,850,752,3,939,350,3,996,345,4,277,437,4,275,149 and 4,366,241, it all incorporates this paper into by reference.

The means that detect described mark are well-known for those skilled in the art.Therefore, for example, can use photographic film or scintillation counter detection of radioactive border note, can use photodetector detection emission light and detect fluorescent mark.Usually by enzyme substrates being provided and detecting by enzyme the reaction product that substrate-function produces is detected enzyme labelling, and there is color marker to detect the color development mark by visual inspection simply.

Can mix described mark by the well-known means of many those skilled in the art.Yet, in a preferred embodiment, during the amplification step of preparation sample nucleic acid, mix described mark simultaneously.Therefore, for example, the polymerase chain reaction (PCR) of using labeled primer or labeled nucleotide will provide the amplified production of mark.In a preferred embodiment, as mentioned above, the transcription amplification of using labeled nucleotide (for example, fluorescein-labeled UTP and/or CTP) mixes mark in the nucleic acid of transcribing.

As an alternative, can directly add mark or after amplification is finished, directly add to original nucleic acid samples (for example, mRNA, poly A mRNA, cDNA etc.) to amplified production.The means of adhering to mark to nucleic acid are well-known for those skilled in the art, for example comprise, nick translation or end mark (for example using the RNA of mark), it is to the nucleic acid effect and with postadhesion (connection) nucleic acid connector by kinases, this connector is connected to mark (for example, fluorophore) with sample nucleic acid.

In a preferred embodiment, by cyanine dyes (for example, Cy-3/Cy-5dUTP, Cy-3/Cy-5dCTP (Amersham Pharmacia)) or alexa dyestuff (Khan, et al., 1998, Cancer Res.58:5009-5013) implementing fluorophore modifies.

In a preferred embodiment, with different fluorescence dyes two target samples that are used for comparison are carried out mark, described fluorescence dye produces recognizable detection signal, for example, and target that makes from normal cartilage with the Cy5 mark and the target that makes from the mild osteoarthritis cartilage with the Cy3 mark.To not the target sample and identical microarray hybridization of isolabeling simultaneously.In a preferred embodiment, use the target of method purifying mark well known in the art, for example by ethanol purification or column purification.

In a preferred embodiment, described target will comprise one or more contrast molecules, and the contrast probe of described molecular hybridization to the microarray is so that the signal standardization that produces from described microarray.Preferably, the stdn target of mark is a nucleotide sequence, and it is complementary to control oligonucleotide fully, and described control oligonucleotide is added on the aforesaid microarray by point.The signal that stdn contrast after hybridization obtains is provided at relevant hybridization conditions, mark intensity, " reading " efficient and can causes the contrast that changes aspect the other factors that complete hybridization signal changes between array.In a preferred embodiment, the signal that all other probes read from array (for example, fluorescence intensity) makes the observed value stdn thus divided by the signal (for example, fluorescence intensity) from described contrast probe.

Select preferred stdn target to be present in the mean length of other target in the sample with reflection, yet, length range has been contained in their selection.The contrast of all right choice criteriaization is with (on average) based composition of other probe in the reflection array, yet, in a preferred embodiment, only use one or several calibrated probe and they are selected so that fully hybridization (that is, do not have secondary structure and do not have self hybridization) and any target molecule that do not match.

Calibrated probe is located in the spatial variations of a plurality of positions of any position in the array or whole array with the control hybridization efficiency.In a preferred embodiment, the stdn contrast is located in angle or the edge and the centre of array.

Hybridization conditions

Nucleic acid hybridization is included in probe or target nucleic acid member target complementary with it and can forms by the complementary base pairing sex change probe or target nucleic acid member and target nucleic acid are provided under the condition of stablizing the heteroduplex body.Wash the nucleic acid that does not form the heteroduplex body subsequently off, stay hybrid nucleic acid to be detected, detect by the detectable label that adheres to usually.It is generally accepted and make nucleic acid denaturation by increasing the salt concn that temperature or reduction contain the nucleic acid damping fluid.(for example, low temperature and/or high salt) will form heteroduplex body (for example, DNA:DNA, RNA:RNA or RNA:DNA) under low rigorous condition, even not be complete complementary situation in the annealed sequence.Therefore, the hybridization specificity reduces under low preciseness condition.On the contrary, (for example, comparatively high temps or than low salt concn) successful hybridization requires less mispairing under higher preciseness condition.

The present invention stipulates hybridization conditions, comprises Dig hybridization mixture (Boehringer); Perhaps,, see and go up and Sambrook et al., see description in going up for example at Ausubel et al. based on the hybridization solution of methane amide.

The method of optimizing hybridization conditions is well-knownly (to see for those skilled in the art, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Vol.24:Hybridization With Nucleic acid Probes, P.Tijssen, ed.Elsevier, N.Y., (1993)).

After the hybridization, remove the mark or the unmarked nucleic acid of not hybridization, can use cleaning easily, on substrate surface, produce the pattern of hybridization target nucleic acid thus from support surface.Many kinds of cleaning solutions are known for those skilled in the art and can be used.Can in variety of way, use the particular detection mode of selecting based on the specific markers of check nucleic acid detect or observe resulting mark, the oligonucleotide of hybridization and/or the crossing pattern of nucleic acid, wherein typical detection means comprises scintillation counting, radioautograph, fluorescence measurement, calorimeter measurement, light emission measurement etc.

Image Acquisition and data analysis

After hybridization and any cleaning step and/or processing subsequently, as mentioned above, detect the crossing pattern that is obtained.Detecting or observing in the crossing pattern, not only detect but also the intensity or the signal value of quantitative mark, its expression will be measured from the signal of each point of hybridization and with it and be compared with the corresponding unit value that sends signal by known quantity end mark target nucleic acid, thereby the counting or the absolute value of each end mark target copy number of specified point on array are hybridized in acquisition with crossing pattern.

Analyzing the method for collecting from hybridizing the data of array is well-known in the art.For example, relate in the fluorescently-labeled situation in the hybridization detection, data analysis can may further comprise the steps: determine fluorescence intensity, it is the function from the substrate location of collected data, remove outlier (that is the data that depart from from the statistical distribution of stipulating) and calculating relative binding affinity from the nucleic acid of checking of remaining data.With the data presentation that obtains is the image with each field strength, and described intensity changes according to bonded oligonucleotide and/or nucleic acid and the binding affinity between the nucleic acid of checking.

Use following detection scheme and analyze two cartilage samples to be compared simultaneously, wherein use each sample of different fluorochrome labels.

First fluorescence color of each composition of scanning microarray.The expression of gene level is proportional described in the fluorescence intensity of each array composition and the sample.

Second fluorescent mark is repeated to implement scanning.The ratio of two fluorescence intensities provides the very accurate and quantitative measurement of gene expression dose relatively in two tissue samples.

In a preferred embodiment, determine the fluorescence intensity of fixed target nucleic acid sequence from image, described image is with being equipped with laser-excitation source and being applicable to that the conventional confocal microscope of the interference filter of Cy3 and Cy5 fluorescent agent gathers.Every kind of fluorescent agent is implemented independent scanning, and wherein each pixel resolution is 225 μ m ²And 65,536 grades of GTGs.Split image identifies that intensity between hybridization zone, two fluorescent agent images of stdn and the standardized mean fluorecence value of calculating about each target are according to Khan, et al., 1998, Cancer Res.58:5009-5013.Chen, et al., 1997, the description of Biomed.Optics 2:364-374.Stdn between the application image be adjusted at two different fluorescent agents carry out mark and it is detected in different efficient.A signal intensity rate that is added in one group of internal control gene on the array by trim point is realized this target.

In another preferred embodiment, scanning array and save as 16 independent tiff images in Cy3 and Cy5 passage.Merge described image and application software and analyze, it comprises that the gridding process is to catch the intensity for hybridization data from each point on the array.Collect the intensity for hybridization of the fluorescence intensity of each point and subtracting background and calculate the Cy5 that measures ratio the Cy3 average intensity.Using linear regression method carries out Cy5 that stdn and supposition measure the slope of the scatter diagram of Cy3 intensity be should be 1.The calculating average ratio also is used for resetting the ratio of data and described slope is adjusted into one.Application is not equal to 1 the expression ratio index as otherness genetic expression.

In an especially preferred embodiment, wherein expect the transcriptional level (and expression thus) of one or more nucleotide sequences in the quantitative sample, the concentration of the mRNA transcript that described target nucleic acid sample is a kind of one or more genes therein or come from the nucleic acid concentration and the proportional nucleic acid samples of described gene transcription level (and expression level thus) of described mRNA transcript.Similarly, the nucleic acid amount of hybridization signal intensity and hybridization is proportional is preferred.Although rigorous relatively proportionality (for example, transcribing ratio doubles to cause mRNA transcript in the sample nucleic acid library to double and hybridization signal doubles) be preferred, the technician it should be understood that described proportionality can be looser and or even nonlinear and significative results still is provided.Therefore, for example, cause in the situation of intensity for hybridization 3-6 times difference in the difference of 5 times of said target mrna concentration, it is enough measuring for most of target.In requiring more accurate quantitatively situation, move suitable contrast to revise the variation of in specimen preparation as described herein and hybridization, introducing.In addition, according to the well-known method of those skilled in the art, use " standard " said target mrna of serial dilution and formulate typical curve.Certainly, to the existence of transcript or lack and carry out in the simply detected situation, do not need complicated contrast or calibration in hope.

For example, if the nucleic acid member on array after the hybridization is not labeled, this shows does not express the gene that comprises described nucleic acid member in the sample in office.If the nucleic acid member is by single dye marker, it shows the gene of only having expressed mark in a sample.Nucleic acid member in the array is just shown in two kinds of samples by two kinds of dye markers and has expressed described gene.Even in each cell, expressed gene once and be detected (1/100th, 000 susceptibility).In two samples to be compared, the difference of expression intensity is the index of differential expression, is not equal to 1.0 at the ratio of intensity described in described two samples, preferably less than 0.7 or greater than 1.2, is more preferably less than 0.5 or greater than 1.5.

RT-PCR

In one aspect of the invention, can be by using reverse transcription (RT) and measuring the expression level of biomarker RNA product of the present invention in conjunction with polymerase chain reaction (PCR) amplification from the RNA product of the biomarker of sample.According to one embodiment of the invention, as skilled in the art to understand, RT can be quantitative.

Be used as template and use biomarker of the present invention and transcribe the Auele Specific Primer of part and start reverse transcription from total RNA of sample or mRNA.It is well-known and at Sambrook et al. that the RNA reverse transcription is become the method for cDNA, and 1989 see description is arranged in going up.Can utilize and to implement primer design from the software (for example, Primer Designer 1.0, Scientific Software etc.) that commerce obtains, the method for the well-known in the art and standard of described software application.

An embodiment of the primer scheme that Application Design and selection the present invention are contained has been described principle and the step that relates to the primer that is designed for PCR in real time and SYBR-Green mensuration.Preferably, this scheme use NCBI (The National Center forBiotechnology Information) (NCBI) search engine and use the PrimerQuest primer-design software.PrimerQuest is based on the software of network, and it is by Integrated DNATechnologies, and Inc. (IDT) develops.This software is based on the Primer3 of Whitehead Institute forBiomedical Research exploitation.

In one embodiment, design the present invention contain primer the time used criterion be that the length of product or amplicon is that 100-150 base, best Tm are preferably 60 ℃ (acceptable scope is 58-62 ℃) and most preferred GC content is 50% (preferable range of 45-55% also is to accept).Helpfully be, avoid 3 base sequences of each primer 3 ' end to be complementary to self or another primer, form thereby reduce " primer-dimer ".Also helpful is, avoid in the primer sequence and primer between complementary sequence.Preferably, avoid at 3 ' 3 of successive of end or a plurality of G or C, and single base repeats greater than 3 bases.Preferably avoid the unbalanced distribution of G/C and A/T enrichment structural domain, and in a specific embodiments, described primer have G or C at 3 ' end.Helpful is that 3 ' end of primer is not T, because 3 ' end has bigger tolerance for the primer of T to mispairing.Also helpful is to avoid mispairing, especially in the 3 ' mispairing of holding; And arrange that at 3 ' end the base of at least 7 uniquenesses is useful.Can also use marking scheme and select the available primer, thereby help to avoid self complementation of selected primer.Therefore for example, specify paired must be divided into+1, mispairing be-1, single pb breach is for-2 and next select the alap primer of score, still in one embodiment, the score of primer is less than 5.Preferably, avoid genome amplification, and similarly, preferred arbitrary primer should be crossed over intron.Preferably, should design primer so that half or the 5 ' end of the Nucleotide of at least 7 primers hybridization to 3 ' end of an exon and remaining hybridization to adjacent exon.In the embodiment of further selecting at, the primer of design is striden exon-exon connexon, thereby distinguishes or prevent the amplification of genome sequence.In another alternate embodiment, thereby the design primer is avoided known SNP.

Can use the primer software program and help the primer that designs and select the present invention to contain, such as " primer is searched software (The Primer Quest software) ", it can obtain by following web site url: biotools.idtdna.com/primerquest/.

When search with when upgrading sequence information from human genome database (Human Genome Database) and being used for the biomarker design of primers, following web site url is an available: 1) NCBILocusLink Homepage: Www.ncbi.nlm.nih.gov/LocusLink/And 2) EnsembleHuman Genome Browser: Www.ensembl.org/Homo sapiens, the biomarker information that advantageous applications is relevant is such as gene or sequence explanation, registration or serial ID, gene symbol, RefSeq# and/or UniGene#.

In case obtained correct target DNA sequence, will produce primer from it, preferably note from LocusLink link or from the exon-intron border of the relevant gene of interest of Ensembl Gene Browser.A preferred means optimizing design of primers is to use three options of BASIC, STANDARD and ADVANCE in the PrimerQuest software.

The BASIC function of advantageous applications PrimerQuest software, under Sequence Information, at first shear and affix to [Sequence] frame, use the PCR in real time parameter setting values and design the PCR primer selectively with title input [Name] frame of primer and with target sequence.Under the standard sequence design conditions, preferred 50 as primer setting return number (Number of Primer Set to Return) and personnel selection is used as the wrong storehouse (Mispriming Library) of causing.In one embodiment, helpfully be, at standard design of primers Premium Features (Advanced Function ofStandard Primer Design) the following option of input down: best primer size: 20 (nt), best primer Tm:60 (℃), best primer GC%:50 (%), product magnitude range: 100-150.In addition, under standard feature, preferably can finely tune following option: primer selection, target, exclusionary zone (ExcludedRegions), inclusion region (Included regions) and initiator codon position.

In case import or selected required parameter, the PrimerQuest that search possibility primer is selected is activated, generation comprises the instrument-mFold of actual sequence, its zero position, length, Tm, GC%, product size penalties and prediction secondary structure to the detailed description of potential forward and reverse primer.Below two standards be the most useful: preferred Δ G should be greater than-3.0kcal.mol ^-1, and preferred Tm should and be not more than 55 ℃ less than 50 ℃.Explain difficulty of scatter diagram, but generally preferably be not chosen in the stain that produces long-diagonal in the scatter diagram, because it very may form hairpin structure.

Preferably, primer should be unique and the pseudogene that do not match for target sequence, and it can confirm by the specificity of using [BLAST] inspection primer.Preferably, can use the OligoAnalyzer that provides by IDTBioTools 3.0 and check the possibility that forms self dimer and heterodimer.Preferably, can use right information and the guide of relevant selection best possibility primer that is provided by IDT BioTools or Primer3, described primer will be to being used for the research of biomarker of the present invention.Preferably, only separate with agarose gel electrophoresis and determine, produce single amplicon and its size and meet and expect that those primers of product are used in the research of described biomarker according to curve analysis.

Below Xiang Guan reference is incorporated this paper: Dieffenbach, C.W., Lowe into by reference, T.M.J., Dveksler, G.S. (1995) General Concepts for PCR Primer Design.In:PCR Primer, A Laboratory Manual (Eds.Dieffenbach, C.W, and Dveksler, G.S.) Cold Spring Harbor Laboratory Press, New York, 133-155, Innis, M.A., and Gelfand, D.H. (1990) Optimization of PCRs.In:PCR protocols, A Guide to Methods and Applications (Eds.Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, TJ.) Academic Press, San Diego, 3-12, Sharrocks, A.D. (1994) The design of primers for PCR.In:PCR Technology, CurrentInnovations (Eds.Griffin, H.G., and Griffin, A.M, Ed.) CRC Press, London, 5-11.

Use the template of reverse transcription product subsequently as PCR.

PCR provides the method for rapid amplifying specific nucleic acid sequence, and this method is used by the catalytic many circulations dna replication dna of archaeal dna polymerase heat-staple, that the rely on DNA interested target sequence that increases.The PCR requirement has nucleic acid to be amplified, is positioned at two single stranded oligonucleotide primers, archaeal dna polymerase, triphosphate deoxyribose nucleotide, damping fluid and salt of sequence side to be amplified.

PCR method is well-known in the art.According to Mullis and Faloona, 1987, Methods Enzymol., PCR is implemented in the description among the 155:335 (it incorporates this paper into by reference).Applying template DNA (1fg, more available 1-1000ng) at least and at least the Oligonucleolide primers of 25pmol implement PCR.Typical reaction mixture comprises: the 10H PCR damping fluid 1 (Perkin-Elmer of the DNA of 2 μ l, the Oligonucleolide primers of 25pmol, 2.5 μ l, Foster City, CA), the Taq archaeal dna polymerase (Perkin-Elmer of 1.25 μ M dNTP, the 0.15 μ l (perhaps 2.5 units) of 0.4 μ l, Foster City, CA) and deionized water, cumulative volume is 25 μ l.Cover mineral oil and use thermal cycler enforcement PCR able to programme.

Require to adjust PCR the circulate duration and the temperature of each step and cycle number according to the preciseness of reality.Determine annealing temperature and selection of time according to the efficient of desired primer and template annealing and the mispairing degree that can tolerate.Those of ordinary skill in the art has the ability of good optimization primer annealing condition preciseness and in its ken.The annealing temperature of using is 30 ℃-72 ℃.The initial sex change of template molecule occurs in 92 ℃-99 ℃, and about 4 minutes, then 20-40 circulation for forming by sex change (94-99 ℃, 15 seconds to 1 minute), annealing (determining temperature according to above argumentation, 1-2 minute) and extension (72 ℃, 1 minute).Final extension step was implemented 4 minutes at 72 ℃ usually, and can be the not timing under 4 ℃ of conditions (0-24 hour) step afterwards.

Can also implement QRT-PCR (it is characterized in that quantitatively) so that the quantitative measurment to gene expression dose to be provided.In QRT-PCR, can in two steps, implement reverse transcription and PCR, perhaps reverse transcription can be implemented simultaneously in conjunction with PCR.Can obtain test kit from commerce ((CA) one of) these technology are implemented with transcribing the specific antisense probe for Perkin Elmer, Foster City such as Taqman for having.This probe has specificity and prepares described probe with the quencher and the fluorescence report probe that are compound in oligonucleotide 5 ' end PCR product (for example, coming from the nucleic acid fragment of gene).Different fluorescent marks is attached to different reporter molecules, and this allows to measure two kinds of products in a reaction.When the Taq archaeal dna polymerase was activated, the fluorescent reporter molecule that this enzyme utilizes its 5 prime excision enzyme activity of 5 ' to 3 ' will be incorporated on the probe of template cut away.In lacking the situation of quencher, described reporter molecules is a fluorescent agent.The amount of change in color and each specific product is proportional and measure by photofluorometer in the reporter molecules, therefore, measures the amount of each color and quantitative PCR product thus.In 96 orifice plates, implement the PCR reaction, so that handle and measure the sample that comes from many individualities simultaneously.The Taqman system has additional advantage, and it does not need gel electrophoresis and allows quantitatively when the application standard curve.

Can be used for using and embed (intercolating) dyestuff, such as QuantiTect SYBR green dye (SYBR Green) PCR (Qiagen, Valencia California) that can obtain from commerce without second technology of electrophoresis detection by quantitative PCR product.The proportional fluorescence of amount of RT-PCR and generation and PCR product is implemented in application as fluorescently-labeled SYBR green dye (it is impregnated in the PCR product during the PCR stage).

After the RNA reverse transcription, can use Taqman and QuantiTect SYBR system.Can in the reaction mixture identical, implement reverse transcription (a step scheme) or before with pcr amplification, implement reverse transcription (two step schemes) earlier with the PCR step.

In addition, be known for other system of quantitative measurment mRNA expression product, comprise Molecular

Its application has the probe of fluorescence molecule and quencher molecule, and described probe can form hairpin structure, makes in the hair clip form, and fluorescence molecule is by cancellation, and when hybridization, the increase of fluorescence provides the quantitative measurment to genetic expression.

Can use and use TaqMan ^TM, SybrGreen, Molecular

Deng QRT-PCR determine the quantitative values of the rna level of reaction amplification.Go up the circulation (Ct value) of measuring fluorescence and determining to reach described threshold level in certain fluorescence threshold level (at least on the background fluorescence level).Therefore, the Ct value is the PCR cycle number that relies on concentration, and the fluorescence of amplified production this moment (amplicon) is on the preset threshold value level.This threshold value can be a fluorescence with respect to background become the level or the more preferably level of index of Response amplification can distinguish the time.For the purpose of the value that is identified for the inputting mathematical model, advantageous applications Δ Ct.The Ct value variable quantity that Δ Ct determines between the product of measured gene (i.e. amplification) and crt gene (being called as house-keeping gene sometimes).As skilled in the art to understand, described crt gene should be the gene that expression level does not change.Typical crt gene is beta-actin or GAPDH.

The technology of other quantitative measurment rna expression includes but not limited to the polymerase chain reaction, ligase chain reaction (LCR), Q β replicative enzyme (is seen, for example, international application No.PCT/US87/00880), isothermal amplification method (is seen, for example, Walker et al. (1992) PNAS 89:382-396), strand displacement amplification reaction (SDA), repair chain reaction, asymmetric quantitative PCR (is seen, for example, the U.S. is No.US200330134307A1 openly) and multiple microballoon pearl mensuration (multiplex microspherebead assay), its description sees Fuja et al., 2004, Journal of Biotechnology108:193-205.

Can measure gene expression dose based on the amplification system of transcribing (TAS) from sample amplification RNA by using, described system comprises amplification of nucleic acid sequences (NASBA) and 3SR.See, for example, Kwoh et al (1989) PNAS USA 86:1173; International open No.WO 88/10315; With U.S. Patent No. 6,329,179.In NASBA, can use conventional phenol/chloroform extracting, thermally denature, lysis buffer is handled and miniature centrifugal (minispin) post DNA isolation and RNA or Guanidinium hydrochloride extract RNA and prepare the nucleic acid that is used to increase.These amplification techniques relate to and will have the primer annealing of target-specific sequence.After polymerization,, and double chain DNA molecule is carried out thermally denature once more with RNase H dna digestion/RNA crossbred.In arbitrary situation, make single stranded DNA become two strands fully by adding the second target-specific primer, then be polymerization.Subsequently, by polysaccharase (such as T7 or SP6) effect, double chain DNA molecule is transcribed exponentially.In the isothermal circulating reaction, the RNA reverse transcription is become double-stranded DNA, and transcribe once with polysaccharase (such as T7 or SP6).Products therefrom, no matter be brachymemma or complete, all show it is the target-specific sequence.

Can use several technology and separate amplified production.For example, can separate the product of amplification, see Sambrook et al., 1989 by agarose, agarose-acrylamide or the polyacrylamide gel electrophoresis of using ordinary method.Can also be according to several technology (see for example PCR Protocols, A Guide to Methods andApplications, Innis et al., Academic Press, Inc.N.Y., (1990)) of the present invention's use without electrophoresis detection by quantitative PCR product.For example, can utilize chromatographic technique to realize separating.The chromatography that has many kinds to use in the present invention: absorption, distribution, ion-exchange and molecular sieve, HPLC and their technical skill of many application, comprise post, paper, thin layer and gas-chromatography (Freifelder, Physical Biochemistry Applications toBiochemistry and Molecular Biology, 2nd ed., Wm.Freeman and Co., NewYork, N.Y., 1982).

Another example of separation method is implemented by the covalent labeling Oligonucleolide primers, and described primer is used in the PCR reaction with all kinds small molecules part.In such separation, on each oligonucleotide, there is different parts.Perhaps be the molecule of antibody or affinity element (if part is a vitamin H), its specificity is used described molecule bag by the surface of plate (such as 96 hole elisa plates) in conjunction with one of described part.The one warp-wise so surface of the plate of preparation applies the PCR reaction, and PCR product just specificity is incorporated into this surface.Cleaning this plate, add the solution that contains second molecule (it is in conjunction with first part) with after removing not binding reagents.This second molecule and certain reporting system interrelate.If the PCR product produces, described second molecule is only in conjunction with described plate, and two kinds of Oligonucleolide primers are impregnated in the PCR end product thus.The amount of detection and quantitative PCR product in commercial plate reader almost similarly detects and the quantitative ELISA reaction subsequently.The system of similar ELISA (such as described here) is developed by Raggio Italgene company, and its commodity are called C-Track.

For the amplification of confirming interested nucleotide sequence must be observed amplified production.A kind of typical observational technique comprises with the ethidium bromide staining gel and under UV light to be observed.As an alternative, if with radioactivity or the overall labeling of fluorescent mark Nucleotide amplified production, can allow the amplified production exposure observe subsequently in the x-radiographic film or under suitable excitation spectrum, separate afterwards.

In one embodiment, realize indirectly observing.After separating amplified production, the nucleic acid probe that makes mark contacts with interested institute amplifying nucleic acid sequence.Preferably, probe preferably with the chromophoric group coupling, but probe can be radiolabeled.In another embodiment, probe and binding partners (such as antibody or vitamin H) coupling, but wherein carry the test section in conjunction with other right member.

In another embodiment, implement to detect by the hybridization of Southern trace and application label probe.The technology that relates to the Southern trace is well-known for those skilled in the art and can finds in many standard books about minute subscheme.See Sambrook et al., 1989, on seeing.Simply, separate amplified production by gel electrophoresis.Contact with film (such as nitrocellulose) with the relief gel, it allows transfer nucleic acid and non-covalent combination.Thereafter, hatch film with chromophoric group coupling probe, this probe can be hybridized with the target amplified production.By the film exposure is implemented to detect in x radiographic film or ion launcher.

An aforesaid example is in U.S. Patent No. 5,279, description arranged in 721, and this patent is by with reference to incorporating this paper into, and it discloses about the equipment of autophoresis and transfer nucleic acid and method.Described equipment allows electrophoresis and the trace without the gel peripheral operation, and is suitable for implementing method of the present invention very ideally.

The nuclease protection is measured

In another embodiment of the present invention, can use the RNA product that nuclease protection is measured (comprising ribonuclease protection assay and S1 nuclease mensuration) detection and quantitative biomarker of the present invention.In the nuclease protection is measured, and antisense probe (have, for example, radio-labeling or heterotope mark) in solution, hybridize with the RNA sample.After the hybridization, probe and RNA strand, not hybridization are degraded by nuclease.Use acrylamide gel and separate remaining protected fragment.Usually, solution hybridization is than more effective based on the hybridization of film, and it can hold the RNA sample of the highest 100 μ g, and the blot hybridization of comparing is up to 20-30 μ g.

Ribonuclease protection assay is the most common type that the nuclease protection is measured, and it requires to use rna probe.In the mensuration that comprises the S1 nuclease, can only use the dna probe of oligonucleotide and other strand.Usually, strand, antisense probe must with the complete homology of target RNA to prevent nuclease cutting probe: the target hybridization body.

The Northern trace

According to conventional Northern hybridization technique known to a person of ordinary skill in the art, Northern trace that can also application standard measures to determine the size of rna transcription thing, identifies the rna transcription thing of alternative splicing and the relative quantity of biomarker RNA product of the present invention.In the Northern trace, at first in the sepharose under the sex change condition via electrophoresis according to size separation RNA sample.Subsequently RNA is transferred to film, crosslinked and hybridization with the probe of mark.The radiolabeled probe of heterotope or high specific acitivity be can use, dna probe, in-vitro transcription rna probe and oligonucleotide random primer, nick translation or that PCR produces comprised.In addition, can use only have portion homologous sequence (for example, from the cDNA of different plant species or genomic DNA fragment, perhaps it comprise exon) as probe.The probe of mark (for example, radiolabeled cDNA, its comprise described dna sequence dna total length, single stranded DNA or fragment) can be random length, be up to few 20, at least 30, at least 50 or at least 100 continuous nucleotides long.Can be by any described probe of many different methods marks that well known to a person skilled in the art.The common tagging that is used for these researchs is radioelement, enzyme, the chemicals of emitting fluorescence and other material when being exposed to UV-light.Many fluorescent materials are arranged is known and can be used as mark.These include but not limited to fluorescein, rhodamine, auramine, Texas is red, AMCA is blue and Lucifer is yellow.A kind of concrete test material is anti-rabbit antibody, and it prepares in goat and through lsothiocyanates coupling fluorescein.Can also or use enzyme labelling protein with radioelement.Can use present any available method of counting and come detection of radioactive labels.Isotopic limiting examples comprises ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁸⁶Re.Enzyme labelling is available equally, and can detect with present any technology of colorimetry, spectrophotometry, spectrophotofluorimetry, amperometry or gasometric method of utilizing.By with the reaction of bridging molecule (such as carbodiimide, vulcabond, glutaraldehyde etc.) with enzyme and selected particle coupling.Can utilize and well known to a person skilled in the art any enzyme.This zymoid example includes but not limited to that peroxidase, beta-D-galactosidase, urase, notatin add peroxidase and alkaline phosphatase.U.S. Patent No. 3,654,090,3,850,752 and 4,016,043 by reference as an example, and they disclose substituting marker material and method.

5.16 measure the technology of biomarker protein of the present invention

Protein

Can utilize standard technique to determine the amount of one or more proteins of interest matter of existing in the sample.For example, can use the amount that standard technique that immunoassay for example connects SDS-PAGE (SDS-PAGE), immunocytochemistry etc. such as for example Western trace, immunoprecipitation is determined one or more proteins of interest matter of existing in the sample of having used.The preferred reagent that detects proteins of interest matter is preferably to have the antibody of detectable label in conjunction with the antibody of proteins of interest matter.

For described detection method, the well-known technology of application those skilled in the art can be separated the protein from sample to be analyzed at an easy rate.For example, the method for protein separation can be according to Harlow and Lane (Harlow, E.and Lane, D., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)) description in.

The preferred method that detects one or more proteins of interest matter comprises by interactional those detection methods of protein-specific antibody.For example, can be according to the description utilization of this paper antibody at proteins of interest matter.Can utilize the well-known technology of those skilled in the art to produce antibody.See, for example, the more detailed argumentation of the described antibody technique of the relevant generation of the 5.2nd joint of the application's 5.19.1 joint and the open No.20040018200 of the U.S., it incorporates this paper into by reference.Briefly, such antibody can be polyclonal, perhaps is more preferably monoclonal.For example, can use the complete antibody or the antibody fragment of conjugated antigen (for example, Fab or F (ab ') 2).Preferably, antibody is the people's or humanized antibody.

For example, can use protein of interest matter is had specific antibody or antibody fragment detects described proteinic existence quantitatively or qualitatively.This can implement by for example immunofluorescence technique.In addition, can use antibody (perhaps its fragment) in histology mode (such as immunofluorescence or immunoelectron microscope) and come in situ detection protein of interest matter.Can implement in situ detection by obtaining histology sample (for example, biopsy specimen) from the patient and using at proteinic traget antibody to it.Preferably use described antibody (or fragment) by the antibody (or fragment) that on biological sample, covers mark.By using such step, not only can determine the existence of proteins of interest matter, can also determine distribution, existence in its cell (for example, chondrocyte and lymphocyte) in sample.Can utilize well-known various Histological method (such as dyeing process), thereby realize described in situ detection.

The immunoassay of proteins of interest matter are generally included with detectable traget antibody hatch biological sample, described antibody can be identified protein of interest matter, and detects binding antibody by many any technology well-known in the art.As following more detailed argumentation, term " mark " can be represented detectable substance is passed through for example coupling (promptly, physical connection) be connected to the direct mark of antibody of antibody, and can represent by with another by direct reactive antibody indirect mark of labelled reagent.The example of indirect labelling comprises that using fluorescently-labeled second antibody detects first antibody.

For example, can make biological sample contact and be fixed in support mutually or carrier such as nitrocellulose, perhaps other can fixed cell, the upholder of cell granulations or soluble protein.Can then use through the fingerprint gene specific antibody of detectable label and handle with the described support phase of suitable buffer solution for cleaning subsequently.Can clean for the second time with damping fluid subsequently and support to remove not binding antibody.Can detect the amount of supporting to go up mutually bonding mark by conventional means subsequently.

In the category of protein agent, " support mutually or carrier " means can conjugated antigen or any upholder of antibody.Well-known upholder or carrier comprise glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, Mierocrystalline cellulose, polyacrylamide, gabbro and magnetite natural or that modify.For purpose of the present invention, described carrier characteristics can be soluble or insoluble to a certain extent.Support material can have any possible structure formation in fact, as long as the link coupled molecule can conjugated antigen or antibody.Therefore, the configuration of upholder can be spherical, and is perhaps cylindrical such as pearl shape, such as the internal surface of test tube, and the perhaps outside surface of bar.As an alternative, described surface can be flat, such as thin slice, testing plate etc.Preferred upholder comprises polystyrene bead.Those skilled in the art should know other many suitable carriers, is used for binding antibody or antigen, perhaps can enough routine tests confirm suitable carriers.

Specific antibody can be it to be connected and to be used for enzyme immunoassay (EIA) (Voller with enzyme by one of mode of detectable label, A., " The Enzyme Linked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2:1-7, Microbiological AssociatesQuarterly Publication, Walkersville, MD); Voller, A.et al, 1978, J.Clin.Pathol.31:507-520; Butler, J.E., 1981, Meth.Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL; Ishikawa, E.et al., (eds.), and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo).Be incorporated into antibody enzyme will with the substrate reactions that is fit to, preferred chromogenic substrate, thus produce by this way can be detected chemical part, for example, detect by spectrophotometry, fluorescent method or vision means.The enzyme that can be used to detect ground mark antibody includes but not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, Δ-5-steroid isomerase, Alcohol Dehydrogenase from Yeast, α-Gan Youlinsuantuoqingmei, triose-phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, notatin, beta-galactosidase enzymes, rnase, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.Can finish described detection with colorimetry, this method is used the chromophoric substrate of enzyme.Can also finish detection by the visual comparison of substrates enzymes level of response and similar preparation standard.

Can also use any method of other many immunoassays and finish detection.For example, utilize radiolabelled antibody or antibody fragment, can (see, for example by application of radiation immunoassay (RIA), Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, it incorporates this paper into by reference) detection protein of interest matter.Can be by or (for example, by radioautography detection of radioactive isotropic substance such as the means of using gamma counter or scintillometer ¹²⁵I, ¹³¹I, ³⁵S or ³H).

Can also use the fluorescent chemicals traget antibody.When fluorescently-labeled antibody is exposed to light time of suitable wavelength, can detect its existence subsequently by fluorescence.In the most frequently used fluorescent mark compound, fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine are arranged.

The metal that can also use emitting fluorescence such as ¹⁵²Eu or other lanthanon can detect ground mark antibody.Can utilize the chelate group (such as diethylene triamine pentacetic acid (DTPA) (DTPA) or ethylenediamine tetraacetic acid (EDTA) (EDTA)) of described metal that these metals are attached to antibody.

Antibody and chemiluminescence compound coupling can also be detected the described antibody of ground mark.Determine the existence of chemiluminescent labeling antibody by detecting the luminous existence that in chemical reaction process, produces subsequently.The chemiluminescent labeling examples for compounds that is particularly useful is luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, theromatic acridinium ester (acridinium ester), imidazoles, acridinium ester salt (acridiniumsalt) and barkite.

Similarly, can applying biological luminophor mark antibody of the present invention.Noclilucence is the chemoluminescence that a class is found in biosystem, and wherein catalytic protein increases the efficient of chemiluminescence reaction.Determine the existence of bioluminescent protein matter by detecting luminous existence.For mark, important bioluminescent compound is fluorescein, luciferase and aequorin (aequorin).

Protein array

The polypeptide of specificity and/or selective binding biomarker protein of the present invention can be fixed on the protein array.Can for example, be used for screening the medical sample (such as bladder body and/or blood) of the polypeptide protein product that has biomarker of the present invention with protein array as diagnostic tool.Described protein array can also comprise antibody and other part, and for example, their combinations are by biomarker encoded polypeptides of the present invention.

For example, at De Wildt et al., 2000, Nature Biotech.18:989-994; Luekinget al., 1999, Anal.Biochem.270:103-111; Ge, 2000, Nuc.Acids Res.28:e3; MacBeath and Schreiber, 2000, Science 289:1760-1763; International open No.WO01/40803 and WO 99/51773A1; With U.S. Patent No. 6,406, the method that produces the polypeptide array has been described in 921.For example, use commercial automated installation, for example Genetic MicroSystems and Affymetrix (Santa Clara, California, USA) or BioRobotics (Cambridge, device UK) add the array of polypeptide with point at a high speed.The substrate of array can be, for example, nitrocellulose, plastics, glass are for example through the glass of surface modification.Described array can also comprise porous matrix, for example acrylamide, agarose or similar polymkeric substance.

For example, described array can be an antibody array, for example, and described in De Wildt (on seeing).Can on filter membrane, cultivate the cell that produces polypeptide ligand with array format.Induce polypeptide to produce, and the antibody of expressing is fixed on the cell position of filter membrane.The information of combination degree on each position of relevant array can be saved as tag file (profile), for example be kept in the Computer Database.

In one embodiment, described array is the protein array, it comprise any amount, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or the arbitrarily combination of biomarker of the present invention most.In another embodiment, array is the protein array, its substantially by any amount of listing in table 1 biomarker, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily biomarker combination and forming most.In another embodiment, array is the protein array, its substantially by any amount of listing in table 1 biomarker, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily biomarker combination most, be included in those of indication in the table 3, and table 2 any amount, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50 biomarker most and form, at least one biomarker of wherein said combination is to be selected from table 1.In another embodiment, array is the protein array, its substantially by any amount of listing in table 4 biomarker, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily biomarker combination and forming most.In another embodiment, array is the protein array, its substantially by any amount of listing in table 4 biomarker, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily biomarker combination most, be included in those of indication in the table 6, and table 5 any amount, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50 biomarker most and form, at least one biomarker of wherein said combination is to be selected from table 4.In another embodiment, array is the protein array, its substantially by any amount of listing in table 7 biomarker, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily biomarker combination and forming most.

Antibody or its Fab are provided in one aspect of the invention, it is incorporated into array, this array is optionally in conjunction with the protein of biomarker of the present invention.

5.17 protein production

Can application standard recombinant nucleic acid method express polypeptide of the present invention or antibody (for example, the protein of biomarker of the present invention).Usually on, with the nucleic acid encoding sequence clone to nucleic acid expression vector.Certainly,, each chain must be cloned into expression vector so if protein comprises multiple polypeptide chain, for example, same or different carrier, it is expressed in same or different cell.If protein is enough little, that is, this protein is less than 50 amino acid whose peptides, just can use the synthetic described protein of automatic methodology of organic synthesis.Give expression to the polypeptide that comprises biomarker of the present invention 5 ' district, 3 ' district or in-line coding district from nucleic acid expression vector, described nucleic acid expression vector only comprises those nucleotide sequences in corresponding biomarker of the present invention 5 ' district, 3 ' district or in-line coding district.Produce the method for antibody, described antibody is at the protein of biomarker of the present invention or at 5 ' district, 3 ' district or in-line coding district encoded polypeptide by biomarker of the present invention.

The expression vector that is used for express polypeptide also may comprise the adjusting sequence except that comprising coded polypeptide or its segmental section, comprise for example promotor, and it is operably connected to interested nucleic acid.A large amount of suitable carriers and promotor are arranged is known for those skilled in the art and can commercial produce recombinant precursor of the present invention.Following carrier is provided by way of example.Bacterium: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, California, USA); PTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden).Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).The preferred library of a preferred classes is a display libraries, and it is described below.

Can use method carrier construction well-known in the art, this carrier comprises polypeptide of the present invention and suitable transcribing/translate control signal.These methods comprise reorganization/genetic recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.For example see, at Sambrook﹠amp; Russell, MolecularCloning:A Laboratory Manual, 3 ^RdEdition, Cold Spring HarborLaboratory, N.Y. (2001) and Ausubel et al, the Current Protocols in MolecularBiology (technology of describing among the Greene Publishing Associates and Wiley Interscience, N.Y. (1989).Can select promoter region from any desired gene of using CAT (CAT) carrier or other carrier with selective marker.Two suitable carriers are pKK232-8 and pCM7.There is the bacterium promotor of specific names to comprise lacI, lacZ, T3, T7, gpt, λ P and trc.Promoter in eukaryote comprises the early stage and late promoter of CMV immediate early promoter, HSV thymidine kinase promotor, SV40, from retroviral LTR promotor, mouse metallothionein(MT)-I promotor and various tissue-specific promoter well known in the art.In some specific embodiments, promotor is an inducible promoter.In other embodiments, promotor is a constitutive promoter.Still in other embodiments, promotor is a tissue-specific promoter.

Usually on, but recombinant expression vector will comprise replication orgin and allow the selective marker of transformed host cell, for example, the ampicillin resistance gene of intestinal bacteria (E.coli) and yeast saccharomyces cerevisiae (S.cerevisiae) nutrient defect type mark (such as URA3, LEU2, HIS3 and TRPl gene), and come from the promotor that cance high-expression gene instructs the downstream configurations sequence to transcribe.Described promotor can be the operon that comes from coding glycolytic ferment (such as glycerol 3-phosphate acid kinase (PGK), the a-factor, acid phosphatase or heat shock protein(HSP) and other).In due course mutually in polynucleotide of the present invention and initial sum terminator sequence and preferably leader sequence (its protein secreting that can instruct translation is to periplasmic space or extracellular medium) be assembled together.Randomly, nucleic acid of the present invention can encoding fusion protein matter, and this protein comprises the terminal identification polypeptide of N-, and it gives desired characteristics, for example, makes the recombinant vectors of expression stable or its purifying is oversimplified.By the polynucleotide of the present invention and translation initiation that is fit to and termination signal being inserted the bacterial expression vector that the reading operated that randomly has functional promotor is built with usefulness mutually in (operable reading phase).Described carrier will comprise one or more can select phenotypic markers and replication orgin, thereby guarantees the maintenance of carrier, and if desired, is provided at the amplification in the host.The prokaryotic organism host who is suitable for transforming comprises that intestinal bacteria, subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium) and Rhodopseudomonas (Pseudomonas), streptomyces (Streptomyces) and staphylococcus (Staphylococcus) belong to each interior kind, and other prokaryotic organism host also can be used as option.

As representational but non-limiting instance, but useful bacterial expression vector can comprise selective marker and come from the bacterium replication orgin of available plasmid on the market, comprise the genetic elements of the cloning vector pBR322 (ATCC 37017) that knows.Described commercial carrier comprises, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega, Madison, Wisconsin, USA).

The invention provides through genetic modification to comprise the host of polynucleotide of the present invention.For example, described host cell can comprise nucleic acid of the present invention, and it is conversion, transfection or the infection method introducing host cell of application of known.The present invention also provides through the host cell of genetic modification with expression polynucleotide of the present invention, and wherein said polynucleotide link to each other with the allogenic adjusting sequence of host cell through operation, and described adjusting sequence drives the expression of polynucleotide in the cell.

The present invention also provides the host cell that contains carrier of the present invention, and wherein the conversion of application of known, transfection or infection method are introduced host cell with nucleic acid.Host cell can be an eukaryotic host cell, such as mammalian cell, eukaryotic host cell such as low, such as yeast cell, perhaps can be prokaryotic cell prokaryocyte, such as bacterial cell.For example, can realize recombinant precursor is introduced host cell by the transfection or the electroporation (Davis, L.et al., Basic Methods in Molecular Biology (1986)) of calcium phosphate transfection, DEAE, dextran mediation.Can also use cell free translation system, use the RNA that comes from DNA construct of the present invention and produce described protein.

Can use any host/vector system and express one or more gene of listing in table 2 or splice variants.Sambrook et al., in Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor, New York (1989) has described suitable clone and the expression vector that is used for protokaryon and eucaryon host, and described document is incorporated this paper with integral body into by reference.Most preferred host cell be the specific polypeptide of undesired expression or with the host cell of the described polypeptide of low-level natural expression.

In a specific embodiments, engineered host cell comprises polynucleotide of the present invention to express endogenous gene can inducing under the regulatory element control, in this case, and can be by the adjusting sequence of homologous recombination replaced endogenous gene.As described herein, can applying gene practice shooting, with separate from heterogeneic adjusting sequence or by the new adjusting sequence sub stituent of genetic engineering method synthetic because of existing regulation domain.Described adjusting sequence can comprise the combination of promotor, enhanser, scaffold attached region, negative regulatory element, transcription initiation site, adjusting protein binding site or described sequence.As an alternative, the sequence of RNA that influence produces or protein structure or stability can be replaced, be removed, be added, perhaps modify by target practice in addition, comprise polyadenylic acid signal, mRNA stability element, splice site, be used to strengthen or improve leader sequence or other change of protein transport or secretion characteristic or improve protein or RNA molecular function or stable sequence.

Host of the present invention can also be yeast or other fungi.In yeast, can use the carrier that contains composing type or inducible promoter in a large number.Relevant discussion is seen Ausubel et al. (eds), Current Protocols in Molecular Biology, Vol.2, Greene Publish.Assoc.﹠amp; Wiley Interscience, Ch.13 (1988); Grant et al., 1987, " Expression andSecretion Vectors for Yeast ", Methods Enzymol.153:516-544; Glover, DNACloning, Vol.II, IRL Press, Wash., D.C., Ch.3 (1986); Bitter, 1987, " Heterologous Gene Expression in Yeast ", Methods Enzymol.152:673-684; With Strathern et al. (eds), The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Press, Vols.I and II (1982).

Potential suitable yeast bacterial strain comprises yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomyces pombe), genus kluyveromyces (Kluyveromyces) bacterial strain, mycocandida (Candida), perhaps can the proteinic yeast strain of expressing heterologous.Potential suitable bacteria bacterial strain comprises intestinal bacteria (Escherichia coli), enterobacteriaceae (enterobacteriaceae) such as serratia marcescens (Serratia marescans), and rod bacterium is such as subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonellatyphimurium), Rhodopseudomonas (Pseudomonads) or can the proteinic any bacterial isolates of expressing heterologous.If in yeast or bacterium, prepare protein, may must modify the protein that is produced, for example pass through the phosphorylation in suitable site or glycosylation with the acquisition functional protein.Chemistry or Enzymology method that can application of known be realized described covalent attachment.

Can utilize various mammalian cell culture systems to come express recombinant protein matter.The example of mammalian expression system comprises that the fibroblastic COS-7 clone of monkey COS cell such as monkey kidney (sees Gluzman, 1981, the description of Cell 23:175 (1981)), Chinese hamster ovary (CHO) cell, people's kidney 293 cell, people's epidermis A431 cell, people Colo205 cell, the 3T3 cell, the CV-1 cell, normal diploid cell, come from the cell strain of external former generation tissue culture, former generation explant (primary explants), the HeLa cell, mouse Lcell, BHK, HL-60, U937, HaK, C127,3T3 or Jurkat cell and other can be expressed the clone of compatible carrier.Mammalian expression vector will comprise replication orgin, suitable promotor and also contain necessary ribosome bind site, polyadenylation site, donor splicing site and acceptor site, transcription termination sequence and 5 ' side non-transcribed sequence.

Can comprise alternate freezing and thawing, sonic treatment, mechanical disruption or application cell cracking agent by the microorganism cells of the broken marking protein of any ordinary method.Usually by at first in cell precipitation, extracting, then step or multistep is saltoutd, water-based ion-exchange or Size Exclusion Chromatograph SEC step are separated in the recombinant polypeptide that produces in the microbial culture.In some embodiments, template nucleic acid is the coded polypeptide label also, for example five or six Histidines.

Can use technical point well known in the art from recombinant protein.For example, Scopes (ProteinPurification:Principles and Practice, Springer-Verlag, New York (1994)) provides many purification of Recombinant (with non-reorganization) proteinic ordinary method.Described method comprises, for example, and ion-exchange chromatography, Size Exclusion Chromatograph SEC, affinity chromatography, selective precipitation, dialysis and hydrophobic interaction chromatography.

The change that those of ordinary skills do technical scheme described herein, modification and other enforcement do not break away from the spirit and scope of the present invention.

Provide following examples, so that invention described herein obtains more comprehensively understanding.Should be understood that these embodiment are only presented for purposes of illustration, and can not by any way it be interpreted as limitation of the present invention.

5.18 identify the side that is used to prevent, treat, control or alleviate the compound of bladder cancer or its symptom Method

5.18.1 identify the method for regulating biomarker expression or active compound

The invention provides the method for authenticating compound, this compound is in conjunction with the product of biomarker of the present invention.The present invention also provides the method for authenticating compound, and this compound is regulated the expression and/or the activity of the product of biomarker of the present invention.The animal model that can be used for researching and developing one or more research bladder cancer via these method compounds identified.In addition, can be used for preventing, treat, control and/or the preventative and therapeutic composition that improves osteoarthritis or its symptom is used as lead compound in exploitation via these method compounds identified.Described method is very useful, because related effort and the huge cost of interior preventive of test potential body and therapeutical agent concentrated on those compounds of identifying by external and stripped method described herein effectively.

The invention provides the method for identifying compound to be tested, wherein check described compound to prevent, treat, control or improve the ability of bladder cancer or its symptom, described method comprises and the following: (a) will express the protein of one or more biomarkers of the present invention or RNA product or its segmental cell of its fragment or one or more biomarkers of expression the present invention and contact with test compound; (b) determine the ability of described test compound in conjunction with described protein, protein fragments, RNA product or RNA part (RNA portion), if compound is in conjunction with described protein, protein fragments, RNA product, RNA part thus, the ability that compound so to be tested prevented, treats, controls or improved bladder cancer or its symptom just obtains confirming.For example, described cell can be the cell in yeast cell or Mammals source.For example, by with described test compound and radio isotope or the coupling of enzyme mark, make and to determine combining of described test compound and described protein, protein fragments, RNA product or RNA part by detecting tagged compound in the conjugate, can realize thus described test compound definite in conjunction with described protein, protein fragments, RNA product or RNA ability partly.For example, can use ¹²⁵I, ³⁵S, ¹⁴C or ³H is the mark test compound directly or indirectly, and comes the detection of radioactive isotropic substance by the direct census radioactive emission or by scintillation counting.As an alternative, can use for example horseradish peroxidase, alkaline phosphatase or luciferase enzymatic labelling test compound, and by determining that being fit to substrate conversion is that product detects the enzyme labelling thing.In a specific embodiments, mensuration comprises, to express protein or its fragment of one or more biomarkers of the present invention or express the RNA product of one or more biomarkers of the present invention or its segmental cell combines described protein with known, protein fragments, the compound contact of RNA product or RNA part, measure mixture thereby form, should measure mixture contacts with test compound, and definite described test compound and described protein, protein fragments, RNA product or the interactional ability of RNA part are wherein determined described test compound and described protein, protein fragments, RNA product or the interactional ability of RNA part comprise determines that described test compound compares with described known compound preferentially in conjunction with described protein, protein fragments, the ability of RNA product or RNA part.

The invention provides the method for identifying compound to be tested, wherein check described compound to prevent, treat, control or improve the ability of bladder cancer or its symptom, described method comprises and the following: (a) protein of one or more biomarkers of the present invention or RNA product or its fragment of its fragment or one or more biomarkers of the present invention are contacted with test compound; (b) determine the ability of described test compound in conjunction with described protein, protein fragments, RNA product or RNA part, if compound is in conjunction with described protein, protein fragments, RNA product, RNA part (RNA portion) thus, the ability that compound so to be tested prevented, treats, controls or improved bladder cancer or its symptom just obtains confirming.Can directly or indirectly determine combining of described test compound and described protein or protein fragments.In a specific embodiments, mensuration comprises, the RNA product of the protein of one or more biomarkers of the present invention or its fragment or one or more biomarkers of the present invention or its fragment are combined described protein with known, protein fragments, the compound contact of RNA product or RNA part, measure mixture thereby form, should measure mixture contacts with test compound, and definite described test compound and described protein, protein fragments, RNA product or the interactional ability of RNA part are wherein determined described test compound and described protein, protein fragments, RNA product or the interactional ability of RNA part comprise determines that described test compound compares with described known compound preferentially in conjunction with described protein, protein fragments, the ability of RNA product or RNA part.Can use the combination between technology confirmed test compound well known in the art and biomarker protein of the present invention or its fragment or biomarker RNA product of the present invention or its part.

In some embodiments of said determination method of the present invention, can be on request fixedly the RNA product of biomarker of the present invention or its part, perhaps its target molecule, thereby be easy to from described RNA product or RNA part, described target molecule or both not complex forms, separate complex form, thereby and adaptation automatic assay.In surpassing in the embodiment of said determination method of the present invention, can be on request the fixedly protein of biomarker of the present invention or its fragment, perhaps its target molecule, thereby be easy to from one or both described proteinic not complex forms, separate complex form, thereby and adaptation automatic assay.Can in being suitable for holding any container of reactant, realize test compound and biomarker protein of the present invention or its segmental combination.The example of container comprises microtiter plate, test tube and micro-centrifuge tube like this.In one embodiment, can provide the fused protein that has added structural domain, described structural domain allows one or both described protein to combine with matrix.For example, glutathione-S-transferase (GST) fused protein can be adsorbed on glutathione agarose gel beads (Sigma Chemical; St.Louis, MO) or by the microtiter plate of gsh derivatize, its subsequently with described test compound, and or combine with the target protein of non-absorption or biomarker protein of the present invention or its fragment, and (for example, under the physiological condition of salt and pH) mixtures incubated under the condition that is of value to mixture formation.After hatching, clean pearl or microtiter plate removing any unconjugated composition, and, directly or indirectly measure mixture and form for example by above-mentioned.As an alternative, described mixture is dissociated from matrix, and can the application standard technology determine that biomarker protein of the present invention or its are segmental in conjunction with level.

Can also in screening assay of the present invention, use other with the technology of proteinaceous solid due to matrix.For example, can utilize the combination of vitamin H and streptavidin to fix protein or its fragment, the perhaps target molecule of biomarker of the present invention.Can use well known technology (for example, biotinylation test kit, Pierce Chemicals; Rockford IL) from the biotinylated protein matter product or the target molecule of vitamin H-NHS (N-hydroxyl-succinimide) preparation biomarker of the present invention, and is fixed in the hole of 96 orifice plates (Pierce Chemical) of the plain bag of strepto-affinity quilt.As an alternative, protein or its fragment to biomarker of the present invention can be responded active antibody derivatize to the hole of plate, and by antibodies with protein capture in the hole.Detect the method for such mixture, except those methods of above-mentioned GST fixed complex, comprise the respond complex compound immunodetection of active antibody of the protein of using with biomarker of the present invention, and the enzyme translocation is fixed, its rely on to biomarker protein of the present invention or its fragment, the perhaps detection of target molecule bonded enzymic activity.

Can also use such as in two hybridization assays or three hybridization assays, (for example seeing U.S. Patent No. 5,283,317 as the protein or the protein fragments of " bait protein matter "; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol.Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; With the open No.WO 94/10300 of international monopoly) determine the interaction of biomarker protein of the present invention or its fragment and test compound or combine.

The invention provides and identify that compound to be tested prevents, treats, controls or improve the method for the ability of osteoarthritis or its symptom, described method comprises: (a) will express the protein of one or more biomarkers of the present invention or the cell of RNA product and contact with test compound; (b) after incubation period, determine the protein of existence in (a) or the amount of RNA product; (c) with the amount in (a) with as yet not with corresponding control cells that described test compound contacts in the amount that exists compare, if therefore the amount in the amount relative comparison of described protein or RNA product changes, the ability that compound so to be tested prevented, treats, controls or improved osteoarthritis or its symptom just is determined.In a specific embodiments, by assay method described herein (for example, microarray or RT-PCR) or assay method well known to those skilled in the art, determine described expression level with respect to the expression level in the contrast changed 5%, 10%, 15%, 25%, 30%, 40%, 50%, 5-25%, 10-30%, at least 1 times, at least 1.5 times, at least 2 times, 4 times, 5 times, 10 times, 25 times, 1-10 doubly or 5-25 doubly.In some alternate embodiment, described method comprises, determine to be present in the biomarker of the present invention of listing in table 1 (comprising those concrete products that table 3 is listed) of described cell or list in arbitrarily at least 2 of table 1 any one or multiple product combination (comprising those concrete products that table 3 is listed) and the biomarker of listing in table 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50,1-5,1-10,5-10,5-25 or 10-40, all or the protein of arbitrarily biomarker combination or the amount of RNA product, and with contrast in the amount of those products compare.

Still in other alternate embodiment, such method comprises, determine to be present in the biomarker of the present invention of listing in table 4 (comprising those concrete products that table 6 is listed) of described cell or list in arbitrarily at least 2 of table 4 any one or multiple product combination (comprising those concrete products that table 6 is listed) and the biomarker of listing in table 5, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50,1-5,1-10,5-10,5-25 or 10-40, all or the protein of arbitrarily biomarker combination or the amount of RNA product, and with contrast in the amount of those products compare.

To transforming to express biomarker of the present invention with technology well known in the art at the cell that utilizes in based on raji cell assay Raji described herein.For example see, U.S. Patent No. 6,245, the III part of " Recombinant Expression Vectors and Host Cells " by name in 527, it is by with reference to incorporating this paper into.As an alternative, can use the cell of endogenous expression biomarker of the present invention.For example, can use the bladder body cell of infinite multiplication.

In a specific embodiments, individual or have an individual isolation of chondrocytes of bladder cancer and/or commitment bladder cancer from " normally ", exist or lacking under the condition of test compound, hatch the different time of described cell (that is, 30 minutes, 1 hour, 5 hours, 24 hours, 48 hours and 96 hours).When screening preventative or therapeutic agent, use the complete sequence clone transfection immortalization bladder body cell of biomarker of the present invention or its functional part.Existing or lacking under the condition of test compound, cultivate the different time (that is, 1,2,3,5,7,10 or 14 day) of cells transfected.After hatching, hybridize from described cell preparation target nucleic acid sample and with probe nucleic acid, described nucleic acid probe is corresponding to the nucleotide sequence of differential expression in the cell of normal, bladder cancer or early stage bladder cancer.For example, use radio-labeling, according to this area and the described nucleic acid probe of well-known method mark described herein.For example,, see and go up or Sambrook et al., on seeing, implement hybridization by the northern trace according to Ausubel et al..According to described herein, on array, target with from the sample of normal individual, with respect to otherness hybridization from bladder cancer or the individual RNA of early stage bladder cancer be index corresponding to the rna expression level of the specific nucleic acid sequence of differential expression cell.As in the result who has incubation step under the described test compound condition, the target sequence changes of expression level is that compound increases or reduce the index that corresponding chondrocyte's specific nucleic acid sequence is expressed.

The present invention also provides and identifies that compound to be tested prevents, treats, controls or improve the method for the ability of bladder cancer or its symptom, and described method comprises: the nucleotide sequence of (a) cell-free extract and code book being invented one or more biomarker protein or RNA product contacts with test compound; (b) determine the protein of existence in (a) or the amount of RNA product; (c) with the amount in (a) with as yet not with corresponding control cells that described test compound contacts in the amount that exists compare, if therefore the amount in the amount relative comparison of described protein or RNA product changes, the ability that compound so to be tested prevented, treats, controls or improved bladder cancer or its symptom just is determined.In a specific embodiments, by assay method described herein (for example, microarray or RT-PCR) or assay method well known to those skilled in the art, determine described expression level with respect to the expression level in the contrast changed 5%, 10%, 15%, 25%, 30%, 40%, 50%, 5-25%, 10-30%, at least 1 times, at least 1.5 times, at least 2 times, 4 times, 5 times, 10 times, 25 times, 1-10 doubly or 5-25 doubly.In some alternate embodiment, described method comprises, determine to be present in described extract biomarker of the present invention at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50,1-5,1-10,5-10,5-25 or 10-40, all or the protein of biomarker combination arbitrarily or the amount of RNA product, and with contrast in the amount of those products compare.

In certain embodiments, determine the amount of the RNA product of biomarker of the present invention, in other embodiments, determine the amount of the labelled protein product of biology of the present invention, and still in other embodiments, determine the amount of the RNA and the protein of biomarker of the present invention.Standard method and the composition of determining biomarker RNA of the present invention or protein amount can be used.Such method and composition is described in detail in the above.

In some specific embodiments, in screening assay described herein, use the amount that test kit is determined biomarker protein of the present invention or RNA product.Such test kit comprises measures biomarker at least 1 of the present invention, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the any amount that all or any biomarker make up, at most at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more protein or required material and the reagent of RNA product expression.In some specific embodiments, test kit can also be included in another or the plurality of reagents of using in the different methods, such as: (1) is from blood or organize the reagent of purifying RNA; (2) produce the primer of checking nucleic acid; (3) dNTP and/or rNTP (premixed or separate), optional dNTP with one or more unique tag and/or rNTP (for example, the dNTP of biotinylated or Cy3 or Cy5 mark); (4) synthesize the back labelled reagent, such as the chemically reactive derivative of fluorescence dye; (5) enzyme is such as reversed transcriptive enzyme, archaeal dna polymerase etc.; (6) various buffer mediums, for example, hybridization and cleaning buffer solution; (7) the probe purified reagent and the component of mark are such as centrifugal post etc.; (8) protein purification reagent; (9) signal generates and detection reagent, for example, and streptavidin-alkaline phosphatase enzyme conjugates, chemiluminescence or chemical luminous substrate etc.In some specific embodiments, test kit comprises the quality control protein of mark in advance and/or rna transcription thing (preferred mRNA) with comparing.

In some embodiments, test kit is the qRT-PCR test kit.In other embodiments, test kit is nucleic acid array and protein array.According to the present invention, such test kit will comprise and nucleic acid member bonded array of the present invention and installation kit thereof at least.As an alternative, can be on array pre-assembled protein of the present invention or nucleic acid member.

In a specific embodiments, the test kit of measuring the RNA product of biomarker of the present invention comprises measure R NA product and expresses necessary material and reagent.For example, can use microarray or RT-PCR test kit and only comprising measure biomarker any amount of the present invention, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 at most, necessary those reagent of RNA product level and the material of all or biomarker combination arbitrarily.As an alternative, in some embodiments, test kit can comprise be not limited to measure biomarker any amount of the present invention, mostly be 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, necessary those materials of RNA product level and the reagent of all or biomarker combination arbitrarily most.For example, the microarray test kit can comprise measurement biomarker any amount of the present invention, mostly be most 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the necessary reagent of RNA product level and the material of all or arbitrarily biomarker combination can also comprise any amount that is different from biomarker of the present invention measured, at most at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more necessary reagent of polygene RNA product level and material.In a specific embodiments, microarray or RT-PCR test kit comprise measurement biomarker any amount of the present invention, at most at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, all or any biomarker make up, it or not any amount of biomarker of the present invention, maximum 1,2,3,4,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,175,200,225,250,300,350,400,450,500,1000,5000,15,000,20,000 or more polygenic, it perhaps not the biological target any amount of the present invention, 1-10,1-100,1-150,1-200,1-300,1-400,1-500,1-1000,25-100,25-200,25-300,25-400,25-500,25-1000,100-150,100-200,100-300,100-400,100-500,100-1000 or 500-1000,1000-5000,5000-10,000,10,000-20,000 or more polygenic necessary reagent of RNA product level and material.

For the nucleic acid microarray test kit, described test kit comprises the probe that is attached to support surface usually.Can come the described probe of mark with detectable label.In a specific embodiments, described probe has specificity to biomarker any amount of the present invention, maximum 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,5 ' district, 3 ' district, in-line coding district, exon, intron, exon connexon or the exon-intron connexon of all or arbitrarily biomarker combination.The microarray test kit can comprise operation instruction that implement to measure and explanation and analysis by the method for implementing the data that described mensuration produces.Test kit can also comprise hybridizing reagent and/or, when the hybridization of probe and target nucleic acid sequence, detect and produce the required reagent of signal.Usually on, the material of microarray test kit and reagent are in one or more containers.Each component of test kit is usually in the container that is fit to himself.

For RT-PCR and/or qRT-PCR test kit, described test kit comprises any amount to biomarker of the present invention, maximum 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50 of preliminary election usually, the specific RNA product (for example, exon, intron, exon connexon and exon-intron connexon) of all or biomarker combination arbitrarily has specific primer.RT-PCR and/or qRT-PCR test kit can also comprise the enzyme (for example, polysaccharase is such as Taq) that is suitable for reverse transcription and/or amplification of nucleic acid and the required deoxynucleotide and the damping fluid of reaction mixture of reverse transcription and amplification.The RT-PCR test kit can also comprise any amount to biomarker of the present invention, maximum 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or arbitrarily the biomarker combination have specific probe.But described probe can be used or need not detection probes (for example, fluorescent mark) carry out mark.Each component of RT-PCR test kit is usually in the container that is fit to himself.Therefore, these test kits comprise the different vessels that is suitable for each reagent, enzyme, primer and probe usually.In addition, the RT-PCR test kit can comprise operation instruction that implement to measure and explanation and analysis by the method for implementing the data that described mensuration produces.

For test kit based on antibody, described test kit can comprise, for example: (1) first antibody (it can be attached to or be not attached to upholder), it is in conjunction with protein of interest matter (for example, biomarker any amount of the present invention, maximum 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, all or any protein of biomarker combination); With, randomly, (2) are different from the second antibody of first antibody, and it is in conjunction with described protein or first antibody and be coupled to detectable label (for example, fluorescent mark, radio isotope or enzyme).Test kit based on antibody can also comprise the pearl of implementing immunoprecipitation.Described each component based on antibody kit is usually in being suitable for the container of himself.Therefore, these test kits comprise the different vessels that is suitable for various antibody usually.In addition, can comprise based on the test kit of antibody and implement the operation instruction measured and explanation and analysis by the method for implementing the data that described mensuration produces.

Also can implement mensuration based on reporter gene with prevention, the treatment of identifying compound to be determined, the ability of handling or alleviate osteoarthritis and symptom thereof.In a specific embodiments, the invention provides prevention, the treatment of identifying compound to be determined, the method for handling or alleviate the ability of osteoarthritis and symptom thereof, described method comprises: (a) with compound and the cells contacting of expressing the reporter gene construct, described construct comprises the reporter gene that the controlling element (for example promotor/enhancer element) with biomarker of the present invention can be operatively connected; (b) expression of the described reporter gene of mensuration; (c) with comparing in the amount in (a) and the corresponding control cells, described control cells does not contact with test compounds, if make the amount of expressed reporter gene change with respect to the amount in the control cells, the ability that compound to be detected prevented, treats, handles or alleviated osteoarthritis and symptom thereof just obtains identifying.According to this embodiment, the described biomarker of expression that described cell can be natural or transform to express described biomarker.In another embodiment, the invention provides and identify that compound to be detected prevents, treats, handles or alleviate the method for the ability of osteoarthritis and symptom thereof, described method comprises: (a) compound is contacted with cell-free extract and reporter gene construct, described construct comprises the reporter gene that the controlling element (for example promotor/enhancer element) with biomarker of the present invention can be operatively connected; (b) expression of the described reporter gene of mensuration; (c) with comparing in the amount in (a) and the corresponding contrast, described contrast does not contact with test compounds, if make the amount of expressed reporter gene change with respect to the amount in the contrast, the ability that compound to be detected prevented, treats, controls or alleviated osteoarthritis and symptom thereof just obtains identifying.

Any reporter gene that well known to a person skilled in the art can be used for the employed reporter gene construct of the inventive method.Reporter gene refers to that coding is easy to detected rna transcription thing or proteinic nucleotide sequence, described detection be by its exist (by, for example, RT-PCR, Northern trace, Western trace, ELISA etc.) or active finishing.The limiting examples of reporter gene is listed in the table 9 hereinafter.Can obtain the nucleotide sequence of reporter gene and definite described element by any method that well known to a person skilled in the art.The nucleotide sequence of reporter gene can, for example, (for example GenBank) obtains from document or in the database.Perhaps, the polynucleotide of coding reporter gene can produce from the nucleic acid from suitable source.If it is unavailable to contain the clone of nucleic acid of coding particular report gene, but described reporter gene sequence is known, encoding the nucleic acid of reporter gene so can chemosynthesis or from suitable source (for example, the cDNA library, perhaps from nucleic acid, the cDNA library that produces among the preferred poly A+RNA, described nucleic acid is to be separated to from any tissue of expressing described reporter gene or cell) obtain by pcr amplification.In case the nucleotide sequence of reporter gene is determined, the nucleotide sequence of described reporter gene can use method well known in the art to operate, with operation nucleotide sequence, recombinant DNA technology for example, rite-directed mutagenesis, PCR etc. (see, for example, described technical description is in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, JohnWiley﹠amp; Sons, NY, it incorporates this paper by reference into integral body), the reporter gene so that generation has the different aminoacids sequence for example produces aminoacid replacement, disappearance and/or insertion.

Table 9: the character of reporter gene and reporter gene product

Reporter gene	Protein active and detection
		CAT (chloramphenicol acetyltransferase)	Shifting the radioactivity ethanoyl detects to paraxin or by thin-layer chromatography and autography
GAL (beta-galactosidase enzymes)	The colourless galactoside of hydrolysis is to produce coloured product
		GUS (beta-glucuronidase)	The colourless glucosiduronate of hydrolysis is to produce coloured product
LUC (luciferase)	Oxyluciferin, luminous
		GFP (green fluorescent protein)	No substrate fluorescin
SEAP (secretor type alkaline phosphatase)	With suitable substrates or with produce chromophoric substrate generation luminous reaction
		HRP (horseradish peroxidase)	When having hydrogen oxide, oxidation 3,3 ', 5,5 '-tetramethyl benzidine is to form coloured complex
AP (alkaline phosphatase)	With suitable substrates or with produce chromophoric substrate generation luminous reaction

According to the present invention, natural or express one or more usually, the cell of the biomarker of the present invention of all or arbitrary combination can be used for method described herein.Perhaps, can use the technological transformation cell that uses in the well known in the art and methods described herein with express any one or multiple, the biomarker of the present invention or the reporter gene of all or arbitrary combination.The example of technology includes but not limited to like this, and calcium phosphate precipitation (see, for example, Graham ﹠amp; Van der Eb, 1978, Virol.52:546), the transfection of the transfection of the transfection of dextran mediation, calcium phosphate mediation, polybrene (polybrene) mediation, protoplastis fusion, electroporation, packing nucleic acid in liposome and directly microinjection nucleic acid in nuclear.

In a specific embodiments, the cell that methods described herein are used is chondrocyte, lymphocyte (T or bone-marrow-derived lymphocyte), monocyte, neutrophilic granulocyte, hugely has a liking for cell, eosinophilic granulocyte, basophilic granulocyte, red corpuscle or thrombocyte.In a preferred embodiment, the cell of methods described herein use is a transitional cell bladder carcinoma cell line.In a further preferred embodiment, the cell of methods described herein use is a lymphocyte.In a further preferred embodiment, the cell of methods described herein use is a white corpuscle.In another preferred embodiment, the cell that methods described herein are used is an all cells in the blood.The cell that methods described herein are used is the immortal cell line that obtains from source (for example tissue).

Any permission translated nucleic acid, optional is still preferred, allows the cell-free extract of transcribed nucleic acid to can be used for method as herein described.Cell-free extract can separate from the cell of any source of species and obtains.For example, the rat cell of the separable mouse cell from human cell, cultivation of cell free translation extract, cultivation, Chinese hamster ovary (CHO) cell, xenopus leavis oocytes, rabbit reticulocyte, Fructus Hordei Germinatus or rye embryo (are seen, for example, Krieg ﹠amp; Melton, 1984, Nature 308:203 and Dignam et al., 1990Methods Enzymol.182:194-203).Perhaps, the cell free translation extract, for example rabbit reticulocyte lysate and malt extract, can available from, for example, Promega, (Madison, WI).In a preferred embodiment, cell-free extract is the extract that separates from the human cell.In a specific embodiments, the human cell is Hela cell, lymphocyte or bladder cell.

Except the ability of the RNA that regulates biomarker of the present invention and/or protein expression level, can expect that also at least some instances, compound is regulated the activity of the protein of biomarker of the present invention.Therefore, the invention provides and identify that compound to be detected prevents, treats, controls or alleviate the method for the ability of bladder cancer and symptom thereof, it comprises the protein activity methods that authenticating compound is regulated the one or more biomarkers of the present invention.Such method can comprise: the cell that (a) will express the protein of the one or more biomarkers of the present invention contacts with test compounds; (b) after incubation period, determine the activity level of protein; (c) more described activity level with not with the contacted corresponding control cells of described test compounds in activity level, make, if the activity level (a) changes with respect to activity level in the control cells, the test compounds ability of preventing, treat, control or alleviate bladder cancer and symptom thereof obtains identifying so.In a specific embodiments, activity level with respect to the activity level in the contrast change the highest by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 5-25%, 10-30%, at least 1 times, at least 1.5 times, at least 2 times, 4 times, 5 times, 10 times, 25 times, 1-10 doubly or 5-25 doubly, as by using mensuration as herein described (for example microarray or RT-PCR) or well known to a person skilled in the art that mensuration is determined.In alternate embodiment, such method comprises, activity level in the contrast is determined be up to few 2 kinds of any amount that exists in the cell, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 12 kinds, at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, the 1-5 kind, the 1-10 kind, the 5-10 kind, the 5-25 kind, or 10-40 kind, the activity level of the protein of the biomarker of the present invention of all or any combination.

The invention provides and identify that compound to be detected prevents, treats, controls or alleviate the method for the ability of bladder cancer and symptom thereof, it comprises: the cell-free extract of coding nucleic acid that (a) will have the protein of the one or more biomarkers of the present invention contacts with test compounds; (b) after incubation period, determine the activity level of protein; (c) more described activity level with not with the contacted corresponding contrast of described test compounds in activity level, make, if the activity level (a) changes with respect to activity level in the contrast, the test compounds ability of prevent, treat, control or alleviate bladder cancer and symptom thereof obtains evaluation so.In a specific embodiments, does activity level change the highest by 1% with respect to the activity level in the contrast? 5%, 10%, 15%, 25%, 30%, 40%, 50%, 5-25%, 10-30%, at least 1 times, at least 1.5 times, at least 2 times, 4 times, 5 times, 10 times, 25 times, 1-10 doubly or 5-25 doubly, as by using mensuration as herein described (for example microarray or qRT-PCR) or well known to a person skilled in the art that mensuration is determined.In alternate embodiment, such method comprises, activity level in the contrast is determined be up to few a kind of any amount that exists in the sample, at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 12 kinds, at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, the 1-5 kind, the 1-10 kind, the 5-10 kind, the 5-25 kind, or 10-40 kind, the activity level of the protein of the biomarker of the present invention of all or any combination.

But the application standard technology is determined the activity level of the protein of biomarker of the present invention.

5.18.2 the biological activity of described compound

Identify compound to be detected prevent, treat, control or alleviates the ability of bladder cancer and symptom thereof after (for the purpose of making things convenient for, this paper is called " guide " compound), can further study described compound.For example, can in acceptable tumour and preferred bladder cancer animal model, carry out the body build-in test in addition by present method compounds identified.In addition, can be to analyzing by the specificity of described method compounds identified.

In one embodiment, the effect that cell growth that can be by measuring target cell or viability are measured lead compound.Such mensuration can use the representative cell of the cell type relevant with bladder cancer (for example bladder stave cell) to implement.Perhaps, can use the cell screening lead compound of clone, to substitute the cell of cultivating from the patient.

Many mensuration well known in the art can be used for assessing contact with lead compound after the survival and/or the growth of patient's cell or clone; For example, cell proliferation can mix by bromodeoxyribouridine (BrdU) (see, Hoshino et al for example, 1986, Int.J.Cancer 38,369; Campana et al., 1988, J.Immunol.Meth.107:79) or ( ³H)-the close pyridine nucleosides of thymus gland mixes and (sees Chen for example, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J.Biol.Chem.270:18367-73), by direct cell counting, by ((for example detecting known such as proto-oncogene, fos, myc) or cell cycle mark (Rb, cdc2, cyclinA, D1, D2, D3, E etc.)) transcribe, translation or active change measure.The level of such protein and RNA (for example mRNA) and activity can be determined by any method well known in the art.For example, can be by known immune diagnostic method (for example using the western trace or the immunoprecipitation of commercially available antibody) to quantification of protein.MRNA can use known in this field and conventional method quantitative, for example, uses northern to analyze, RNase protection, the polymerase chain reaction relevant with reverse transcription.Cell viability can expect that blue dyeing or other necrocytosis known in the art or survival mark assesses by using platform.In a specific embodiments, measure the cell ATP level to determine cell viability.Can assess differentiation, for example, visually assess based on morphologic change.

Animal model

Compound can be tested in suitable animal model system before human body is used.Such animal model system includes but not limited to rat, mouse, chicken, ox, monkey, pig, dog, rabbit etc.Can use any animal system well known in the art.In certain embodiments, test compounds in mouse model.But repetitive administration compound.

The animal model of generally acknowledging can be used for detecting the effectiveness that is used to prevent, treat, control and/or improve bladder cancer or its symptom by aforesaid method institute compounds identified.

Toxicity

The toxicity of compounds identified and/or effectiveness can be measured in cell culture or laboratory animal according to the medicinal program of standard according to the present invention, for example, measure LD ₅₀(making 50% lethal dosage in the colony) and ED ₅₀(to 50% medicable dosage in the colony).Can be used for evaluating the Cytotoxic cell and the clone of compounds identified, include but not limited to, peripheral blood lymphocytes (PBMC), Caco-2 cell and Huh7 cell according to the present invention.Dosage ratio between toxicity and the curative effect is a therapeutic index, and it can be expressed as LD ₅₀/ ED ₅₀Compounds identified is preferred according to the present invention to demonstrate big treatment exponential.Although can use the compounds identified that demonstrates toxic side effect according to the present invention, but design delivery system that should be careful, it so that the latent lesion of non-infected cells is minimized, and reduces side effect with the tissue site of such its effect of medicament target thus.

From cell cultures measure and zooscopy the data that obtain can be used for systematically discussing the dosage range that according to the present invention compounds identified is used for human body.The dosage of such medicament preferably is in the circulation composition scope, and it comprises seldom toxicity or does not have toxic ED ₅₀According to formulation that is adopted and route of administration, described dosage can change in this scope.For any medicament that uses in the method for the present invention, can from measuring, cell cultures infer the treatment effective dose at first.Can systematically discuss dosage in animal model, to obtain the circulating plasma concentration range, it is included in the IC that measures in the cell cultures ₅₀(that is, reach half of the maximum inhibition of symptom compound concentration).Such information can be used for measuring more accurately the useful dosage in the human body.For example, can pass through the high-performance liquid chromatogram determination blood plasma level.

Homologue or analogue design

The compound that demonstrates required biologic activity can be used as lead compound and is used for exploitation or designs to have active homologue of beneficial drugs or analogue.For example, in case identify lead compound, can adopt the molecule modeling technique to design the variant of described compound, it can be more effective.The example of molecule modeling have CHARM and QUANTA program (Polygen Corporation, Waltham, MA).CHARM carries out energy minimization and molecule power mathematic(al) function.QUANTA carries out structure, graphical modeling and analysis of the molecular structure.QUANTA allows interactive mode structure, modification, visual and molecular behavior analysis to each other.

The microcomputer modelling with the interactional medicine of specific protein summarized in many articles, Rotivinen et al. for example, 1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, 1998, New Scientist 54-57; McKinaly ﹠amp; Rossmann, 1989, Annu.Rev.Pharmacol.Toxiciol.29:111-122; Perry ﹠amp; Davies, and OSAR:QuantitativeStructure-Activity Relationships in Drug Design pp.189-193 (Alan R.Liss, Inc.1989); Lewis ﹠amp; Dean, 1989, Proc.R.Soc.Lond.236:125-140and141-162; Askew et al., 1989, J.Am.Chem.Soc.111:1082-1090., other screening and the graphical computer program of describing chemical substance are also arranged, it is from a plurality of companies, BioDesign for example, Inc. (Pasadena, California), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario) although. these mainly are to be designed for specified protein is had specific medicine, can make them be applicable to that design has specific medicine to any zone of identifying.Can use above-mentioned screening, described analogue and homologue are detected with combining of proteins of interest matter (being the protein product of biomarker of the present invention) at biologic activity.As an alternative, seldom or do not have a lead compound of biologic activity, as described in the screening find, also can be used for designing the analogue and the homologue of compound with biologic activity.

5.18.3 compound

Can include but not limited to according to methods described herein detection and compounds identified, compound from any commercial source acquisition, comprise Aldrich (1001 West St.Paul Ave., Milwaukee, WI 53233), (P.O.Box 14508 for Sigma Chemical, St.Louis, MO 63178), (Industriestrasse 25 for FlukaChemie AG, CH-9471Buchs, Switzerland (FlukaChemical Corp.980South 2nd Street, Ronkonkoma, NY 11779)), EastmanChemical Company, Fine Chemicals (P.O Box 431, Kingsport, TN 37662), Boehringer Mannhcim GmbH (Sandhofer Strasse 116, D-68298Mannheim), Takasago (4Volvo Drive, Rockleigh, NJ 07647), SSTCorporation (635Brighton Road, Clifton, NJ 07012), Ferro (LA 70791 for 111West IreneRoad, Zachary), Riedel-deHaen Aktiengesellschaft (P.O.BoxD-30918, Seelze, Germany), PPG Industries Inc., Fine Chemicals (OnePPG Place, 34th Floor, Pittsburgh, PA 15272).In addition, can use the natural product of method screening any kind of of the present invention, it comprises bacterium, fungi, plant or animal extracts.

Can screen coming arrogant compound synthetic or the natural compounds library.At present, several different methods is used at random with orientation synthetic based on sugar, based on peptide with based on the compound of nucleic acid.The synthetic compound library is commercially available, and it comprises Maybridge Chemical Co. (Trevillet, Cornwall from a plurality of companies, UK), and Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT).Rare chemical substance library is an available, its from Aldrich (Milwaukee, WI).Combinatorial library is available and has prepared.As an alternative, the natural compounds library that exists with the form of bacterium, fungi, plant and animal extract is an available, (Bothell WA) or MycoSearch (NC), perhaps is easy to produce according to method well known in the art from for example Pan Laboratories for it.In addition, natural and synthetic library and compound are easy to modify by traditional chemical, physics and biochemical method.

In addition, can utilize diversified test compounds library, it comprises little analytical test compound.Use the library of the inventive method screening can comprise polytype compound.Example according to the library of the inventive method screening includes but not limited to the class peptide; Biological at random oligomer; Various body (diversomers) is hydantoins, benzene phenodiazine for example

Class and dipeptide; Vinylogous polypeptide (vinylogous polypeptides); Non-peptide type is intended peptide class (nonpeptidalpeptidomimetics); The oligomerization amino formate; Peptide phosphonic acid ester (peptidylphosphonates); The peptide nucleic acid(PNA) library; Antibody library; The sugar library; And small molecules library (preferred little organic molecule library).In some embodiments, the compound in the described screened library is nucleic acid or peptide molecule.In a non-restrictive example, peptide molecule can be present in the phage display library.In other embodiments, the kind of compound includes but not limited to, peptide analogs, it comprises the peptide that contains alpha-non-natural amino acid (for example D-amino acid), amino acid whose phosphoramidate analog, for example alpha-amino group phosphoric acid and alpha-amino group phosphoric acid, perhaps has the amino acid that non-peptide connects, nucleic acid analog, for example group thiophosphate (phosphorothioates) and PNA class, hormone, antigen, synthetic or natural drug, opiates, Dopamine HCL, serotonin, catecholamines, zymoplasm, vagusstoff, prostanoid, organic molecule, the pheromone class, adenosine, sucrose, glucose, lactose and semi-lactosi.Polypeptide or protein library also can be used for mensuration of the present invention.

In a specific embodiments, described combinatorial library is little organic molecule library, and it includes but not limited to, the benzene phenodiazine

Class, isoprenoid, thiazolidine ketone (thiazolidinones), metathiazanones, Pyrrolidine class (pyrrolidines), morpholino compounds and benzene phenodiazine

Class. in another embodiment, described combinatorial library contains the class peptide; Biological at random oligomer; The benzene phenodiazine

Class; Various body is such as hydantoins, benzene phenodiazine

Class and dipeptide; Vinylogous polypeptide; Non-peptide type is intended the peptide class; The oligomerization amino formate; The peptide phosphonic acid ester; The peptide nucleic acid(PNA) library; Antibody library; Or sugared library.Combinatorial library self is commercially available.For example, the library can commercial obtain, and it is from for example, Specs and BioSpecs B.V. (Rijswijk, The Netherlands), ChembridgeCorporation (San Diego, CA), Contract Service Company (Dolgoprudny, Moscow Region, Russia), and Comgenex USA Inc. (Princeton, NJ), MaybridgeChemicals Ltd. (Cornwall PL34OHW, United Kingdom), Asinex (Moscow, Russia), ComGenex (Princeton, New Jersey), Ru, Tripos, Inc. (St.Louis, Missouri), ChemStar, Ltd (Moscow, Russia), and 3D Pharmaceuticals (Exton, Pennsylvania), with Martek Biosciences (Columbia, Maryland).

In a preferred embodiment, described library is selected in advance, makes the compound in described library be suitable for the cell absorption more.For example, select compound based on special parameter, described parameter such as but not limited to, size, lipotropy, wetting ability and hydrogen bond, it has increased the easy degree that compound enters cell.In another embodiment, by three-dimensional or four-dimensional computer calculates program described compound is analyzed.

Can synthesize according to the combination of compounds library of the inventive method being used for.Produce a large amount of little organic molecule set or the synthetic method in library has very big interest for orientation, can screen pharmacy, biology or other activity in wherein said set or library.Carry out in order to the described synthetic method (promptly on carrier) in solution or in mutually of creating big combinatorial library.Solid phase synthesis makes the control polystep reaction be more prone to, and drives reaction and finish with high yield, because excessive reactant can add easily and flush away after per step reaction.The solid phase combination is synthesized and is also helped to improve separation, purifying and screening.But more traditional solution phase chemistry is than the more organic reaction of solid state chemistry support.

U.S. Patent No. 6,190 can be used in combination of compounds of the present invention library, and the described device of 619 (Kilcoin etc.) synthesizes, and it incorporates this paper into by reference in its entirety.U.S. Patent No. 6,190,619 disclose a kind of synthesizer, and it can support a large amount of reaction vessels, to be used for the parallel synthetic of multiple isolated compound or to be used for the combination of compounds library.

In one embodiment, the combination of compounds library can be synthesized in solution.U.S. Patent No. 6,194,612 (Boger etc.) disclosed method makes compound be used as the template that combinatorial library solution is combined to, it incorporates this paper into by reference in its entirety.The design of described template be make to allow use liquid/liquid or solid-liquid extract with reaction product easily from unreacted reactant purifying come out.To be preferably little organic molecule by the synthetic composition that produces of the combination of using described template.Some compounds in the library can be simulated the effect of non-peptide or peptide.With respect to the solid phase synthesis in combination of compounds library, liquid phase is synthesized does not need to use special scheme, with each step (Egner et al., 1995, the J.Org.Chem.60:2652 in the monitoring multistep solid phase synthesis; Anderson et al., 1995, J.Org.Chem.60:2650; Fitch et al., 1994, J.Org.Chem.59:7955; Look et al., 1994, J.Org.Chem.49:7588; Metzger et al., 1993, Angew.Chem., Int.Ed.Engl.32:894; Youngquist et al., 1994, Rapid Commun.Mass Spect.8:77; Chu et al., 1995, J.Am.Chem.Soc.117:5419; Brummel et al., 1994, Science 264:399; AndStevanovic et al., 1993, Bioorg.Med.Chem.Lett.3:431).

The combination of compounds library that is used for the inventive method can be synthesized on solid support.In one embodiment, disperse synthesis method (split synthesis method), in building-up process, separate and mix the scheme of upholder, be used for synthetic compound library on solid support (see, for example, Lam et al., 1997, Chem.Rev.97:41-448; Ohlmeyer et al., 1993, Proc.Natl.Acad.Sci.USA 90:10922-10926, and the document of wherein being quoted).Each solid support in final library all has a kind of compound in its surface attached basically.The method of other synthetic combinatorial libraries on solid support will be that those skilled in the art are known, one of them product be attached on each upholder (see, Nefzi et al. for example, 1997, Chem.Rev.97:449-472).

In some embodiments of the present invention, compound is attached on the solid support by connexon.Connexon can be to be incorporated on the solid support and be its part that perhaps connexon can be nonconformity, synthesizes on solid support or attached to it in synthetic back.Connexon not only is used to provide compound to be connected to the tie point of solid support, and is used for making not molecule on the same group to cut down from solid support under different condition according to the character of connexon.For example, connexon can be, the electrophilic cutting, and the nucleophilic cutting, but the light cutting, the enzyme cutting, metal cutting, under reduction or oxidizing condition, cut.In a preferred embodiment, described compound was cut from solid support before high flux screening.

If the array or the microarray of described library inclusion compound, wherein every kind of compound has an address or sign, and described compound can be resolved, and for example is assigned to the precursor compound tabulation of using by positive is intersected in each is measured.

If described library is peptide or nucleic acid library, the sequence of described compound can be measured by the direct order-checking to peptide or nucleic acid.Such method is well known by persons skilled in the art.

Many physical-chemical technology can be used for the sign again of compound.The example of such technology includes but not limited to, mass spectrum, NMR spectrum, X ray crystalline diffraction and vibrational spectrum.

5.19 institute's compounds identified is applied to prevent, treat, control or alleviate bladder cancer or its symptom

The invention provides prevention, treat, control or alleviate the method for bladder cancer or its symptom, described method comprises one or more is administered to required object according to the inventive method institute compounds identified.In a preferred embodiment, described human to liking.

In one embodiment, the invention provides prevention, treat, control or alleviate the method for bladder cancer or its symptom, described method comprise the prevention or the treatment significant quantity one or more be administered to required object according to the inventive method institute compounds identified.In a specific embodiments,, do not use to prevent, to treat, to control or to alleviate bladder cancer or its symptom according to the inventive method compounds identified if such compound once was used for preventing, treat, control or alleviating bladder cancer or its symptom in the past.In another embodiment, if such compound once made the people expect can be used for preventing, treat, control or alleviating bladder cancer or its symptom, do not use to prevent, to treat, to control or to alleviate bladder cancer or its symptom according to the inventive method compounds identified.In another embodiment, only the protein of a biomarker or RNA product combine and/or change its expression level and/or activity level with the present invention specifically according to the inventive method compounds identified.In another embodiment, (be up to few 2, at least 3, at least 4 according to the inventive method compounds identified and any amount of the present invention, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 or more) protein of biomarker or RNA product combine and/or change its expression level and/or activity level.

In a specific embodiments, increase or reduce the assimilation and/or the alienation activity of the transitional cell bladder carcinoma cell line of bladder cell or infinite multiplication according to the inventive method compounds identified.Preferably, compare with the transitional cell bladder carcinoma cell line of untreated bladder cell or infinite multiplication, such compound assimilates the chondrocyte and/or the alienation activity increases or reduces greater than 1 times, and more preferably, 1.5-5 times, most preferably, 5-100 doubly.In another embodiment, alleviate at least a symptom relevant and/or change according to the inventive method compounds identified, comprising the symptom of bladder cancer with bladder cancer.In a specific embodiments, using with the prevention or the therapeutical agent that prevent, treat, control or alleviate bladder cancer or its symptom is synthetic compound or natural product (for example plant milk extract or culture supernatant), or the mixture of compound.

The present invention also provides prevention, treats, controls or alleviates the method for bladder cancer or its symptom, described method comprises uses above-mentioned screening method institute compounds identified to be administered to required object one or more, and one or more other treatments (for example prevention or therapeutical agent and operation).In a specific embodiments, current such treatment of using be used for preventing, treat, control or alleviate bladder cancer or its symptom (including but not limited to hereinafter listed prevention or the therapeutical agent of part) or known be useful to it.Treatment in the combined therapy of the present invention (for example prevention or therapeutical agent) can be used or use simultaneously in proper order.In a specific embodiments, combined therapy of the present invention comprises compounds identified other treatment identical with at least a and described compound effects mechanism according to the present invention.In another particular, combined therapy of the present invention comprises the composition of identifying according to the inventive method, and different other treatment (for example, prevention or therapeutical agent) of at least a and described compound effects mechanism.Combined therapy of the present invention has additional or synergism by concuring with described compound, has improved the prevention or the result of treatment of The compounds of this invention.Combined therapy of the present invention has reduced and the relevant side effect of described treatment (for example prevention or therapeutical agent).

The prevention of combined therapy or therapeutical agent can be administered to object in identical medicinal compositions.As an alternative, the prevention of combined therapy or therapeutical agent can independently be administered to object in the medicinal compositions respectively simultaneously.Prevention or therapeutical agent can be administered to object by identical or different route of administration.

In a specific embodiments, the medicinal compositions that will contain one or more compounds of identifying in the mensuration described herein is administered to object, and is preferred human, to prevent, to treat, to control or to alleviate bladder cancer or its symptom.According to the present invention, described medicinal compositions also can comprise one or more preventions or therapeutical agent.Preferably, such medicament is to be applied at present, to be applied to or knownly be applied to preventing, treat, control or alleviating bladder cancer or its symptom.

The method according to this invention compounds identified can be used as a line, two wires of bladder cancer, three-way, four lines or the treatment of five lines.For the object of traditional bladder cancer treatment tolerance, the invention provides treatment, control or alleviate the method for bladder cancer or its symptom.Described method comprise use prevention or treatment significant quantity give described object according to the inventive method compounds identified.

Object for existing single bladder cancer pharmaceutical treatment tolerance, the invention provides treatment, control or alleviate the method for bladder cancer or its symptom, described method comprises that one or more other treatments according to the inventive method compounds identified and prevention or treatment significant quantity (for example prevention or therapeutical agent) of using prevention or treatment significant quantity give described object.The present invention also provides the method for treatment or control bladder cancer, and it will be by giving the patient who has confirmed other treatment is tolerated and do not re-use these treatments according to the inventive method compounds identified and other treatment arbitrarily (for example operation) combined administration.The present invention is also to suffering from bladder cancer and providing the method for the treatment of or controlling owing to immunosuppressant patient appears in other treatment of accepting before.The present invention has been proved for the object of receiving treatment or controlling in hormonotherapy and/or biotherapy/immunotherapy and maybe may be proved under the toxicity situation of excessive (promptly causing unacceptable or insufferable side effect), and the alternative method of treatment or control bladder cancer also is provided.

5.19.1 be used to prevent, treat, control or alleviate the compound of bladder cancer or its symptom

Can be used for the method according to this invention describes in detail hereinafter with the representative non-limitative example that prevents, treats, controls and/or alleviate the compound of bladder cancer or its symptom.

At first, such compound can comprise, for example, can reduce the protein of biomarker of the present invention or expression or active antisense, ribozyme or the triple helical compound of RNA product.Such compound has a detailed description hereinafter.

Secondly, such compound can comprise, for example, the protein of adjustable abridged edition invention biomarker or RNA product are expressed or the active antibody compositions of protein of biomarker of the present invention.In a specific embodiments, protein or the expression of RNA product, the perhaps activity of the protein of biomarker of the present invention of described antibody compositions downward modulation biomarker of the present invention.Such compound has a detailed description hereinafter.

The 3rd, such compound can comprise, for example, and the protein product of biomarker of the present invention.The present invention includes selected peptide or peptide mimics and be used to simulate the protein of biomarker of the present invention, to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In addition, such compound can comprise, and for example, can regulate protein or the expression of RNA product or the negative polypeptide (dominant-negativepolypeptides) of the active dominance of biomarker protein of the present invention of biomarker of the present invention.

Described method comprises that also derivative, analogue or the fragment of biomarker protein of the present invention are in the application that prevents, treats, controls or alleviate on bladder cancer or its symptom.Especially, the present invention includes the fragment of biomarker protein of the present invention that to comprise one or more structural domains of this proteinoid and be applied to prevention, treat, control or alleviate bladder cancer or its symptom.In another embodiment, the present invention includes the protein of biomarker of the present invention or be expressed as and merge or proteinic like this analogue, derivative or the segmental application of chimeric protein product (comprising the protein, fragment, analogue or the derivative that are connected to the heterologous protein sequence by peptide bond).

In a specific embodiments, the antisense oligonucleotide of using at least a kind, at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, at least 10 kinds, at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds or more kinds of biomarkers of the present invention is to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In another embodiment, use the protein of one or more biomarkers of the present invention or its fragment, analogue or derivative to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In other embodiments, use one or more and protein specificity bonded antibody of the present invention to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In other embodiments, use the negative polypeptide of one or more dominance to prevent, to treat, to control or to alleviate bladder cancer or its symptom.

Antisense, ribozyme, triple helical composition

But the application standard technology is to produce antisense, triple helical or ribozyme molecule, with the part as methods described herein.At first, but the application standard technology produces antisense nucleic acid molecule, promptly with the positive phosphorothioate odn complementation of coding polypeptide of interest, for example, with the coding strand of double-stranded cDNA molecule complementary or with the complementation of mRNA sequence.Therefore, antisense nucleic acid can be connected with positive phosphorothioate odn hydrogen bond.Described antisense nucleic acid can be complementary or only complementary with its part with whole coding strand, for example, and all or part protein coding region (or opening code-reading frame).Antisense nucleic acid molecule can be the antisense nucleic acid of all or part non-coding region on the coding strand of nucleotide sequence of coding polypeptide of interest.Non-coding region (" 5 ' and 3 ' non-translational region ") is 5 ' and the 3 ' zone adjacent with coding region, and does not translate into amino acid.

Antisense oligonucleotide can be, for example, on the length up to the Nucleotide of about 5,10,15,20,25,30,35,40,45 or 50 or more any amount.Antisense nucleic acid of the present invention can use chemosynthesis and enzyme ligation (using step known in the art) to make up.For example, antisense nucleic acid (as antisense oligonucleotide) can use the Nucleotide of naturally occurring Nucleotide or multiple modification to carry out chemosynthesis, the design of described modified nucleotide is for the biologically stable that increases described molecule or increases antisense and the physical stability of the duplex that positive phosphorothioate odn forms, the Nucleotide that for example can use phosphorothioate derivative and acridine to replace.The example that can be used for producing the modified nucleotide of antisense nucleic acid comprises 5 FU 5 fluorouracil (5-fluorouracil), 5-bromouracil (5-bromouracil), 5-chlorouracil (5-chlorouracil), 5-iodouracil (5-iodouracil), xanthoglobulin (hypoxanthine), xanthine (xanthine), the close pyridines of 4-acetyl born of the same parents (4-acetylcytosine), 5-(carboxymethyl) uridylic (5-(carboxyhydroxylmethyl) uracil), 5-carboxymethylamino methyl-2-sulfo-uridine (5-carboxymethylaminomethyl-2-thiouridine), 5-carboxymethyl aminomethyl uridylic (5-carboxymethylaminomethyluracil), dihydrouracil (dihydrouracil), β-D-semi-lactosi Q nucleosides (beta-D-galactosylqueosine), Trophicardyl (inosine), N6-isopentenyl gland purine (N6-isopentenyladenine), 1-methyl guanine (1-methylguanine), 1-methyl inosine (1-methylinosine), 2,2-dimethylguanine (2,2-dimethylguanine), 2-methyladenine (2-methyladenine), 2-methyl guanine (2-methylguanine), 3-methylcystein (3-methylcytosine), 5-methylcytosine (5-methylcytosine), N6-VITAMIN B4 (N6-adenine), 7-methyl guanine (7-methylguanine), 5-methyl aminomethyl uridylic (5-methylaminomethyluracil), 5-methoxyl group aminomethyl-2-thiouracil (5-methoxyaminomethyl-2-thiouracil), β-D-seminose Q nucleosides (beta-D-mannosylqueosine), 5 '-methoxyl group carboxymethyl uracil (5 '-methoxycarboxymethyluracil), 5-methoxyuracil (5-methoxyuracil), 2-methylthio group-N6-isopentenyl gland purine (2-methylthio-N6-isopentenyladenine), uridylic-5-ethoxyacetic acid (v) (uracil-5-oxyacetic acid (v)), bosom fourth glycosides (wybutoxosine), pseudouracil (pseudouracil), Q nucleosides (queosine), 2-sulphur cytosine(Cyt) (2-thiocytosine), 5-methyl-2 thiouracil (5-methyl-2-thiouracil), 2-thiouracil (2-thiouracil), 4-thiouracil (4-thiouracil), methyl uracil (5-methyluracil), uridylic-5-ethoxyacetic acid methyl esters (uracil-5-oxyacetic acid methylester), uridylic-5-ethoxyacetic acid (v) (uracil-5-oxyacetic acid (v)), 5-methyl-2-thiouracil (5-methyl-2-thiouracil), 3-(3-amino-3-N-2-carboxylic propyl group) uridylic (3-(3-amino-3-N-2-carboxypropyl) uracil), (acp3) w, with 2, the 6-diaminopurine (2,6-diaminopurine).As an alternative, can use with antisense orientation (being that the RNA that the transcribed nucleic acid that inserted produces will be the antisense orientation of interested target nucleic acid) subclone the expression of nucleic acids carrier to produce antisense nucleic acid in the biology mode.

Antisense nucleic acid molecule is administered to object or produces in position, makes them with the cell mRNA hybridization of coding polypeptide of interest or combine, and suppresses thus to express, and for example, transcribes and/or translates by inhibition.Described hybridization can be stablized duplex and finishes to form by the complementation of traditional core thuja acid, perhaps, for example, with the situation of DNA duplex bonded antisense nucleic acid molecule under, interact by the specificity in the duplex major groove.The example of the route of administration of antisense nucleic acid molecule of the present invention is included in tissue place direct injection, for example, and joint (such as knee joint, hip joint, elbow joint and articulations digitorum manus).As an alternative, can modify with the selected cell of target antisense nucleic acid molecule, general is used then.For example, for general is used, can modify antisense molecule, make acceptor or the antigen of their specificitys in conjunction with selected cell (T cell or chondrocyte) surface expression, described combination for example is connected to polypeptide or antibody by described antisense nucleic acid molecule, and described polypeptide or antibody combine with cell surface receptor or antigen.Described antisense nucleic acid molecule also can be delivered to cell by using carrier (for example gene therapy vector hereinafter described).For reaching enough ICs of described antisense molecule, it is preferred that antisense nucleic acid molecule position vector construction body thereon is under the control of strong polII or polIII promotor.

Interested antisense nucleic acid molecule can be α-end group isomery (anomeric) nucleic acid molecule.α-end group isomery nucleic acid molecule and complementary RNA form specific double-strand hybridization, and be wherein opposite with common α-unit, be parallel to each other between the chain (Gaultier etc., 1987, Nucleic Acids Res.15:6625-6641).Antisense nucleic acid molecule also can comprise 2 '-o-methyl ribonucleotides (2 '-o-methylribonucleotide) (Inoue etc., 1987, Nucleic Acids Res.15:6131-6148) or chimeric RNA-DNA analogue (Inoue etc., 1987, FEBS Lett.215:327-330).

Ribozyme is the catalytic RNA molecule with ribonuclease activity, and it can cut the single-chain nucleic acid that has with its complementary region, and such as mRNA, and it also can use standard technique to produce.Therefore, ribozyme (for example hammerhead ribozyme (as Haselhoff and Gerlach, 1988, Nature334:585-591 is described)) can be used for catalytic cutting mRNA transcript, and suppresses the translation of described mRNA coded protein thus.The nucleic acid molecule that has for the coding polypeptide of interest has specific ribozyme to design based on cDNA nucleotide sequence disclosed herein.For example, the derivative of TetrahymenaL-19IVS RNA can be fabricated the wherein nucleotide sequence of avtive spot and nucleotide sequence complementation to be cut in the U.S. Patent No. 5,116,742 of the U.S. Patent No. 4,987,071 of Cech etc. and Cech etc.As an alternative, the mRNA of coding polypeptide of interest can be used for the catalytic RNA that selection has the specific ribonucleic acid enzymic activity from the RNA library of molecules.See, for example, Barteland Szostak, 1993, Science 261:1411-1418.

Triple-helix structure also can use known technology to generate.For example, the expression of polypeptide of interest can be suppressed by the targeted nucleotide sequence, itself and regulation and control zone (for example promotor and/or the enhanser) complementation of the encoding gene of the polypeptide that forms triple-helix structure, and described structure stops described gene transcribing in target cell.Usually, visible Helene, 1991, Anticancer Drug Des.6 (6): 569-84; Helene, 1992, Ann.NY.Acad.Sci.660:27-36; And Maher, 1992, Bioassays14 (12): 807-15.

In multiple embodiments, nucleic acid composition can be modified at base portion, sugar moieties or phosphoric acid skeleton, to improve for example stability, crossability or the solvability of molecule.For example, the deoxyribose phosphate skeleton of nucleic acid can be modified with generate peptide nucleic acid(PNA) (see Hyrup etc., 1996, Bioorganic﹠amp; Medicinal Chemistry 4 (1): 5-23).As used herein, term " peptide nucleic acid(PNA) " or " PNA " refer to nucleic acid mimics, for example, dna analog, wherein the deoxyribose phosphate skeleton is replaced by pseudopeptide backbone, and has only four kinds of nucleic acid bases to be retained.The neutral backbone of PNA has demonstrated the specific hybrid of permission DNA and RNA under conditions of low ionic strength.The synthetic available standards solid-phase peptide synthetic schemes of PNA oligomer is implemented, and it is as Hyrup etc., 1996, supra; Perry-O ' Keefe etc., 1996, described in the Proc.Natl.Acad.Sci.USA 93:14670-675.

For example, can be by additional lipotropy or other auxiliary group on PNA, by forming the PNA-DNA mosaic, or PNA is modified by liposome or other medicine delivery technique known in the art, take in such as stability that strengthens them or cell.For example, the PNA-DNA mosaic can be generated, and it combines the advantage of PNA and DNA character.Such mosaic allows DNA identification enzyme, and for example, RNAase H and archaeal dna polymerase partly interact with its DNA, and its PNA part will provide high avidity and specificity.The PNA-DNA mosaic can use the connexon of appropriate length to be connected, its length according to the number and the direction of key between base stacking, the nucleic acid base select (Hyrup, 1996, as above).PNA-DNA chimeric synthetic can be as Hyrup, 1996, as above and Finn etc., 1996, Nucleic Acids Res.24 (17): 3357-63 is described to be implemented.For example, can use standard phosphoramidite connection chemistry and modified nucleoside analog on solid support, the DNA chain to be synthesized.Compound for example 5 '-(4-methoxyl group trityl) amino-5 '-deoxidation-thymus pyrimidine phosphoramidite can be used as connection (Mag etc., 1989, Nucleic Acids Res.17:5973-88) between the 5 ' end of PNA and DNA.Then, the PNA monomer is connected in mode one by one, forms chimeric molecule (Finn etc., 1996, Nucleic Acids Res.24 (17): 3357-63) with 5 ' PNA fragment and 3 ' dna fragmentation.As an alternative, chimeric molecule can use 5 ' dna fragmentation and 3 ' PNA fragment to synthesize (Peterser etc., 1975, Bioorganic Med.Chem.Lett.5:1119-11124).

In other embodiments, oligonucleotide can comprise other additional group, such as peptide (for example be body in target host cell receptor), perhaps promote cross-cell membrane (to see, Letsinger etc. for example, 1989, Proc.Natl.Acad.Sci.USA 86:6553-6556; Lemaitre etc., 1987, Proc.Natl.Acad.Sci.USA 84:648-652; The world is No.WO 88/09810 openly)) or stride the reagent (seeing that for example, the world is No.WO89/10134 openly) that hemato encephalic barrier is transported.In addition, oligonucleotide can use the cutting agent that hybridization causes (see, for example, Krol etc., 1988, Bio/Techniques6:958-976) or intercalating agent (see, for example, Zon, 1988, Pharm.Res.5:539-549) modify.For this purpose, oligonucleotide can combine with another molecule (for example, the cutting agent that causes of the linking agent, transport agents, the hybridization that cause of peptide, hybridization etc.).

Antibody compositions

In one embodiment, will be administered to object with one or more protein specificity bonded antibody of the one or more biomarkers of the present invention, preferred human, in order to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In another embodiment, will give object with any combined administration of one or more protein specificity bonded antibody of the one or more biomarkers of the present invention, preferred human, in order to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In a specific embodiments, to be administered to object with one or more one or more antibody of protein specificity bonded of the one or more biomarkers of the present invention, preferred human, in conjunction with the treatment of other type in order to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In a specific embodiments, be administered to object with known in the art with the one or more protein specificity bonded antibody one or more biomarkers of the present invention, preferred human, separately or in conjunction with the treatment of other type in order to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In other embodiments, be not administered to object with the one or more protein specificity bonded antibody one or more biomarkers of the present invention with known in the art, preferred human, separately or in conjunction with the treatment of other type in order to prevent, to treat, to control or to alleviate bladder cancer or its symptom.

Use multiple delivery system well known by persons skilled in the art, will be administered to object with one or more one or more antibody of protein specificity bonded of the one or more biomarkers of the present invention, preferred human.For example, such antibody can be used by being packaged in liposome, particulate or the micro-capsule.See, for example, U.S. Patent No. 5,762,904, U.S. Patent No. 6,004,534 and international open No.WO 99/52563.In addition, such antibody can be used by the retrovirus, other virus vector or the non-virus carrier that use reconstitution cell that can expressing antibodies or can express described antibody.

Can obtain from any known source with one or more protein specificity bonded antibody of the one or more biomarkers of the present invention.Perhaps, can make by any currently known methods of this area synthetic antibody, particularly or preferably, pass through recombination and expression techniques by chemosynthesis with one or more protein specificity bonded antibody of the one or more biomarkers of the present invention.

Antibody includes but not limited to, polyclonal antibody, monoclonal antibody, bi-specific antibody, multi-specificity antibody, people's antibody, humanized antibody, camelization (camelised) antibody, chimeric antibody, strand Fvs (scFv) (see, for example (1988) Science 242:423-426 such as Bird; With (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883 such as Huston), single-chain antibody, single domain antibody, Fab fragment, F (ab ') fragment, the disulfide linkage Fvs (sdFv), antiidiotype (anti-Id) antibody that are connected (comprises, for example, at the antiidiotypic antibody of antibody of the present invention) and above antigen arbitrarily in conjunction with and/or the epi-position binding fragment.Term used herein " antibody " refers to the immunocompetence fragment of immunoglobulin molecules and immunoglobulin molecules, promptly contains the molecule of antigen binding site.Immunoglobulin molecules can be any kind (for example, IgG, IgE, IgM, IgD, IgA and IgY), classification (for example, IgG ₁, IgG ₂, IgG ₃, IgG ₄, IgA ₁, IgA ₂) or subclass.The segmental example of the immunocompetence of immunoglobulin molecules comprises F (ab) fragment (the unit price fragment of being made up of VL, VH, CL and CH1 structural domain) and F (ab ') ₂Fragment (containing two segmental divalence fragments of Fab that link to each other by disulfide linkage at hinge area), it can produce by using the enzyme processing antibody such as stomach en-or papoid.The immunocompetence fragment includes but not limited to that also Fd fragment (being made up of VH and CH1 structural domain), Fv fragment (VL and VH structural domain by the antibody single armed are formed), dAb fragment (are made up of the VH structural domain; Ward etc., (1989) Nature 341:544-546) and isolating complementary determining region (CDR).The antibody of specificity conjugated antigen can make by any currently known methods of this area synthetic antibody, particularly by chemosynthesis or preferably, passes through recombination and expression techniques.

The polyclonal antibody of specificity conjugated antigen can make according to several different methods well known in the art.For example, the human antigen can be administered to multiple host animal, and it includes but not limited to, rabbit, mouse, rat etc. produce the serum that contains human antigen's specific polyclonal antibody to induce.Multiple adjuvant can be used for increasing immunne response, it depends on host type, include but not limited to that freund's adjuvant (fully and not exclusively), mineral coagulant be for example lysolecithin, pluronic polyol class (pluronic polyols), polyanion class, peptide, oil-emulsion, key hole limpet hemocyanin (keyhole limpet hemocyanins), dinitrophenol and may be useful in human adjuvant for example BCG (bacille Calmette-Guerin vaccine) and Acne (corynebacterium parvum) of aluminium hydroxide, tensio-active agent for example.Such adjuvant also is well known in the art.

Term " monospecific antibody " refers to show for the single binding specificity of particular target (for example epi-position) and the antibody of affinity.This term comprises monoclonal antibody.Monoclonal antibody can be used many technology preparations known in the art, and it comprises hybridoma, reorganization and display technique of bacteriophage, or its combination.See that for example, U.S. Patent No. RE 32,011,4,902,614,4,543,439,4,411,993and 4,196, and 265; Kennett etc. (eds.), Monoclonal Antibodies, Hybridomas:A New Dimension in Biological Analyses, Plenum Press (1980); With Harlow and Lane (eds.), Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), it is incorporated herein by reference.For example, monoclonal antibody can use hybridoma technology to produce, and it is known in the art and for example that described technology comprises, Harlow etc., Antibodies:A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed.1988); Hammerling, etc., in:MonoclonalAntibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981) instructed in (described reference is incorporated this paper by reference into integral body).Can produce other technology (for example, William D.Huse etc., 1989, Science, the 246:1275-1281 of antibody by recombinant technology; L.Sastry etc., 1989, Proc.Natl.Acad.Sci.USA, 86:5728-5732; And MichelleAlting-Mees etc., Strategies in Molecular Biology, 3:1-9 (1990) involving acommercial system available from Stratacyte, La Jolla, the described technology of Calif) also can be applicable to make up monoclonal antibody.Term as used herein " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation.Term " monoclonal antibody " refers to that described clone comprises any eukaryotic cell, prokaryotic cell prokaryocyte or phage clone, rather than refers to the method that it produces from the antibody of single clone's generation.

It is this area routine and known using the method for hybridoma technology generation and screening specific antibody.In brief, can use the protein immune mouse of mark of the present invention,, for example in mice serum, detect the antibody of described protein-specific, gather in the crops spleen and the separating Morr. cell of described mouse in case detect immunne response.Then by known technology the myeloma cell of splenocyte and any appropriate is merged, for example from the cell of SP20 clone, it can derive from ATCC.Select and the clone hybridization knurl by limiting dilution.In addition, RIMMS (multidigit point repeat immunological technique) can be used for immune animal (Kilptrack etc., 1997, Hybridoma 16:381-9, it incorporates this paper into by reference in its entirety).Measure the hybridoma clone by methods known in the art then, can be to obtain secretion in conjunction with the cell of the antibody of polypeptide of the present invention.By using positive hybridoma clone immune mouse, produce the ascites that contains high-level antibody usually.

Therefore, the invention provides method by the hybridoma producing antibody of cultivating secretion antibody of the present invention, wherein, preferably, described hybridoma produces by merging splenocyte (it separates from the protein mice immunized of using biomarker of the present invention) and myeloma cell, then the hybridoma of gained is merged in screening, can be in conjunction with the hybridoma clone of the antibody of described protein or protein fragments to obtain secretion.

The antibody fragment of the protein specificity epitope of identification biomarker of the present invention can produce by any technology well known by persons skilled in the art.For example, Fab of the present invention and F (ab ') ₂Fragment can make by the proteolysis cutting of immunoglobulin molecules, and it for example uses papoid (producing the Fab fragment) or stomach en-(produce F (ab ') ₂Fragment) enzyme.F (ab ') ₂Fragment contains the CH1 structural domain of variable region, constant region of light chain and heavy chain.In addition, antibody of the present invention also can use multiple phage display method known in the art to produce.

In the phage display method, the functional antibodies structural domain is illustrated in the surface of phage particle, and described particle carries their polymerized nucleoside acid sequence of coding.Especially, the amplification of the dna sequence dna of coding VH and VL structural domain is from animal cDNA library (for example, the people of infected tissue or mouse cDNA library).The DNA of coding VH and VL structural domain interconnects with the scFv connexon by PCR, and the clone enters the phagemid carrier.Described carrier electroporation enters E.coli and E.coli is infected by helper phage.The phage of using in these methods is filobactivirus normally, and it comprises fd and M13, and VH merges with phage gene III or gene VIII with recombination form usually mutually with the VL structural domain.Can use antigen to select with the phage of specific antigen bonded antigen binding domains or identify expressing, the antigen of applying marking for example perhaps is connected with solid surface or pearl or by its antigen of catching.The example that can be used for the phage display method of production antibody of the present invention comprises following discloses: Brinkman etc., 1995, J.Immunol.Methods 182:41-50; Ames etc., 1995, J.Immunol.Methods 184:177-186; Kettleborough etc., 1994, Eur.J.Immunol.24:952-958; Persic etc., 1997, Gene 187:9-18; Burton etc., 1994, Advances in Immunology 57:191-280; PCT applies for PCT/GB91/O1 134; International open WO 90/02809, WO 91/10737, and WO 92/01047, and WO 92/18619, and WO 93/11236, and WO 95/15982, and WO 95/20401, and WO97/13844; And United States Patent (USP) 5,698,426,5,223,409,5,403,484,5,580,717,5,427,908,5,750,753,5,821,047,5,571,698,5,427,908,5,516,637,5,780,225,5,658,727,5,733,743 and 5,969,108, more than each incorporates this paper into integral body as a reference.

As described in above reference, after phage is selected, can be separated and be used to produce whole antibody from the antibody coding zone of phage, it comprises human antibodies, or any other required Fab, and express in any required host, described host comprises mammalian cell, insect cell, vegetable cell, yeast and bacterium, and is for example as described below.Also can utilize the reorganization of using means known in the art to produce Fab, Fab ' and F (ab ') ₂Segmental technology, such as following discloses, international open WO 92/22324; Mullinax etc., 1992, BioTechniques 12 (6): 864-869; Sawai etc., 1995, AJRI 34:26-34; And Better etc., 1988, Science240:1041-1043 (described reference is incorporated this paper by reference into integral body).

Be to produce complete antibody, can use PCR primer amplification VH or VL sequence in the scFv clone, the flanking sequence that described primer comprises VH or VL nucleotide sequence, restriction site and is used to protect restriction site.Use clone technology well known by persons skilled in the art, the VH structural domain of described pcr amplification can be cloned the into carrier of expression VH constant region (for example people γ 4 constant regions), and the VL structural domain of described pcr amplification is cloned the into carrier of expression VL constant region (for example people κ or λ constant region).Preferably, the carrier of expression VH or VL structural domain comprises EF-1 α promotor, secretion signal, variable domains cloning site, constant domain and selective marker (for example Xin Meisu).Described VH and VL structural domain also can be cloned the same carrier of the necessary constant region of into expression.Then, use technology well known by persons skilled in the art, heavy chain is changed carrier (heavy chainconversion vectors) and light chain change the carrier cotransfection and enter clone, to produce the clone of stable or transient expression full length antibody (for example IgG).

For some application, comprise antibody in intravital application of people and vitro detection mensuration, end user's antibody or chimeric antibody may be preferred.People's antibody is to expect especially for the therapeutic treatment of human subjects fully.People's antibody can be prepared by several different methods known in the art, and it comprises that use mentioned above derives from the phage display method of the antibody library of human normal immunoglobulin sequence.Also can referring to, United States Patent (USP), 4,444,887 and 4,716,111; And international open WO98/46645, WO 98/50433, and WO 98/24893, WO98/16654, WO 96/34096, WO96/33735 and WO 91/10741; More than each by with reference to incorporating this paper into integral body.

Antibody also can produce by transgenic animal.Especially, people's antibody can use transgenic mice to produce, and described transgenic mice can not the endogenous immunoglobulin (Ig) of expressive function, but but its expressing human immunoglobulin gene.For example, people's heavy chain and light chain immunoglobulin gene complex body can import at random or import mouse embryo stem cell by homologous recombination.Perhaps, except that described people's heavy chain and light chain gene, people variable region, constant region and diversity district (diversity region) can import mouse embryo stem cell.Importing along with the human immunoglobulin gene's seat that passes through homologous recombination can make described murine heavy chain and light chain immunoglobulin gene lose function respectively or simultaneously.Especially, J _HThe homozygous deletion in district stops endogenous production of antibodies.The increase embryonic stem cell of described modification and microinjection enters blastocyst to produce gomphosis mouse.Then cultivate gomphosis mouse to produce the offspring of isozygotying of expressing human antibody.Use selected antigen (polypeptide for example of the present invention in whole or in part) with the described transgenic mice of common form immunity.Use conventional hybridization knurl technology, can be from be obtained anti-described antigenic antibody by the transgenic mice of immunity.In the process of B cytodifferentiation, the contained human normal immunoglobulin transgenosis of transgenic mice is reset, and then experiences type conversion and somatic mutation.Therefore, use such technology, might produce IgG, the IgA, IgM and the IgE antibody that can be used for treating.Summary about this technology of producing people's antibody, as seen, Lonberg and Huszar (1995, Int.Rev.Immunol.13:65-93). for the going through of technology that produces people's antibody and human monoclonal antibodies and produce the scheme of such antibody, as seen, for example international open WO 98/24893, WO 96/34096, and WO96/33735; And United States Patent (USP) 5,413,923,5,625,126,5,633,425,5,569,825,5,661,016,5,545,806,5,814,318 and 5,939,598, it incorporates this paper by reference into integral body.In addition, and company (for example, Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA)) also participates in providing anti-selected antigenic people's antibody, and it uses and above describes similar technology.

For example, United States Patent (USP) 5,849,992 have described the method in the mammary gland expressing antibodies of transgenic mice.Making up transgenosis makes it comprise the nucleic acid and the secretory signal sequence of newborn specificity promoter and coding antibody interested.The breast that such female transgenic mammal produces comprises, and secretes in antibody interested wherein.Described antibody can come out from the Ruzhong purifying, perhaps directly uses in some applications.

Chimeric antibody is the molecule of the different piece of described antibody from different immunoglobulin molecules.The method that produces chimeric antibody is known in the art.See, for example, Morrison, 1985, Science229:1202; Oi etc., 1986, BioTechniques 4:214; Gillies etc., 1989, J.Immunol.Methods 125:191-202; With United States Patent (USP) 5,807,715,4,816,567,4,816,397 and 6,331,415, it incorporates this paper by reference into integral body.

Humanized antibody is can be in conjunction with antibody or its variant or the fragment of predetermined antigens, and it comprises framework region that has the human normal immunoglobulin aminoacid sequence basically and the CDR that has the non-human immunoglobulin aminoacid sequence basically.Humanized antibody comprise basically following all, at least one and common two variable domains (Fab, Fab ', F (ab ') ₂Fabc, Fv), wherein all or basically all CDR districts corresponding to non-human immunoglobulin (being donor antibody), and all or basically all framework regions are concensus sequences of human normal immunoglobulin.Preferably, humanized antibody also comprises at least a portion constant region for immunoglobulin (Fc), is typically human normal immunoglobulin.Usually, described antibody will contain the light chain and the variable domains of heavy chain at least.Described antibody also can comprise CH1, hinge, CH2, CH3 and the CH4 district of heavy chain.Described humanized antibody can be selected from any immunoglobulin class, comprises IgM, IgG, IgD, IgA and IgE, and any hypotype, comprises IgG ₁, IgG ₂, IgG ₃And IgG ₄Usually, described constant domain is the constant domain of complement fixation(CF), and when expecting that humanized antibody shows cytotoxicity, classification is typically IgG1.When such cytotoxicity is not expected, constant domain can be the IgG2 class.Described humanized antibody can comprise from more than a type or isostructural sequence, and to select the particular constant structural domain be the ordinary skill of this area to optimize effector function.The framework region of humanized antibody and CDR district do not need accurately corresponding to auxiliary sequence, for example, donor CDR or consistent framework, described CDR or framework can be by replacing, inserting or lack at least one residue and suddenlyd change, and make described CDR or framework residue not correspond to consistent antibody in this site or import antibody.But such sudden change will not be general.Usually, at least 75% humanized antibody residue will be corresponding to female FR and CDR sequence, and more frequent is 90%, most preferably is greater than 95%.Humanized antibody can use multiple technology known in the art to produce, and described technology includes but not limited to that CDR transplants (European patent EP 239,400; International open WO 91/09967; With United States Patent (USP) 5,225,539,5,530,101 and 5,585,089), frosting (veneering) or come to the surface again (resurfacing) (European patent EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka etc., 1994, ProteinEngineering 7 (6): 805-814; With Roguska etc., 1994, PNAS 91:969-973), strand displacement (United States Patent (USP) 5,565,332), and the technology of following discloses, for example, United States Patent (USP) 6,407,213, United States Patent (USP) 5,766,886, WO 9317105, Tan etc., 2002, J.Immunol.169:1119-25, Caldas etc., 2000, Protein Eng.13 (5): 353-60, Morea etc., 2000, Methods 20 (3): 267-79, Baca etc., 1997, J.Biol.Chem.272 (16): 10678-84, Roguska etc., 1996, Protein Eng.9 (10): 895～904, Couto etc., 1995, CancerRes.55 (23Supp): 5973s-5977s, Couto etc., 1995, Cancer Res.55 (8): 1717-22, Sandhu JS, 1994, Gene 150 (2): 409-10, with Pedersen etc., 1994, J.Mol.Biol.235 (3): 959-73.Frequent, the framework residue in the framework region will be replaced from the corresponding residue of CDR donor antibody, to change preferred raising, antigen combination.These frameworks are replaced and are identified by techniques well known, for example, by the interaction modeling of described CDR and framework residue being identified to antigen, and relatively identify extraordinary framework residue at specific position by sequence in conjunction with important framework residue.(as seen, for example, Queen et al., U.S. Patent No. 5,585,089; With Riechmann et al., 1988, Nature 332:323, it incorporates this paper by reference into integral body.)

Single domain antibody (antibody that for example, lacks light chain) can produce by means commonly known in the art.See Riechmann etc. 1999, J.Immuno.231:25-38; Nuttall etc., 2000, Curr.Pharm.Biotechnol.1 (3): 253-263; Muylderman, 2001, J.Biotechnol.74 (4): 277302; United States Patent (USP) 6,005,079; With the open WO 94/04678 in the world, WO94/25591 and WO 01/44301, wherein each incorporates this paper by reference into integral body.

In addition, the antibody of specificity conjugated antigen can then be applied to produce antiidiotypic antibody, and its use well known to a person skilled in the art technology " simulation " antigen.(as seen, for example, Greenspan﹠amp; Bona, 1989, FASEB is (5) J.7: 437-444; And Nissinoff, 1991, J.Immunol.147 (8): 2429-2438).Such antibody can use separately, or is used in combination with other therapies, to prevent, to treat, to control or to alleviate bladder cancer or its symptom.

The present invention includes polynucleotide, it comprises antibody or its segmental nucleotide sequence of coding specificity conjugated antigen.The present invention be also included within high rigorous, in or under the low rigorous hybridization conditions with the polynucleotide of the polymerized nucleoside acid hybridization of code book invention antibody.

Described polynucleotide and determine the nucleotide sequence of described polynucleotide to obtain by any method known in the art.The nucleotide sequence of coding known antibodies can use method well known in the art to determine, that is, the method assembling that the Nucleotide codon of known coded specific amino acids can be such is to produce nucleic acid encoding said antibody.The polynucleotide of such encoding said antibody can be from the oligonucleotide of chemosynthesis (for example, as Kutmeier etc., 1994, BioTechniques 17:242 is described) assembling, it briefly, comprise synthetic overlapping oligonucleotide, fragment or its variant that contains the partial sequence of encoding antibody, anneal and connect these oligonucleotides, the oligonucleotide that connects by pcr amplification then.

Perhaps, the polynucleotide of encoding antibody can produce from the nucleic acid from suitable source.If it is unavailable to contain the clone of nucleic acid of the specific antibodies of encoding, but the sequence of described antibody molecule is known, the nucleic acid of coding immunoglobulin (Ig) can chemosynthesis or from suitable source (for example, antibody cDNA library or generation are from the cDNA library of the tissue or the cell of any this antibody of expression, or separation is from the nucleic acid of above-mentioned tissue or cell, preferred poly A+RNA, described tissue or cell are such as the hybridoma of selected expression antibody of the present invention) obtain by pcr amplification or clone, described pcr amplification used can with the synthetic primer of 3 ' and 5 ' end sequence hybridization, described clone has used has specific oligonucleotide probe to specific gene sequence, described gene order is be to identify, for example from the cDNA clone in the cDNA library of encoding said antibody.The amplification of nucleic acid that produces by PCR can then use any method well known in the art to be cloned into reproducible cloning vector.

In case the nucleotide sequence of described antibody is determined, can use this area the nucleotide sequence of described antibody to be handled as the known method of process nuclear nucleotide sequence, for example recombinant DNA technology, site-directed mutagenesis, PCR etc. are (as seen for described method, for example, Sambrook etc., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, NY and Ausubel etc., eds., 1998, Current Protocols inMolecular Biology, John Wiley﹠amp; Sons, the technology described in the NY, it incorporates this paper by reference into integral body), with the antibody that generation has the different aminoacids sequence, for example produce aminoacid replacement, disappearance and/or insertion.

In case it is (preferred to have obtained encoding antibody molecule of the present invention, heavy chain of antibody or light chain or its fragment, but it is not necessary, contain heavy chain or light chain variable structural domain) polynucleotide, the carrier that produces described antibody molecule can use technology well known in the art to produce by recombinant DNA technology.

In a preferred embodiment, in mammalian cell, produce monoclonal antibody.The mammalian host cell that preferably is used to express described clonal antibody or its Fab comprises that Chinese hamster ovary (Chinese hamster ovary celI) (comprises the dhfr-CHO cell, it is described in Urlaub andChasin (1980, Proc.Natl.Acad.Sci.USA 77:4216-4220), it has used the DHFR selective marker, for example, as Kaufman and Sharp (1982, Mol.Biol.159:601-621) described in, lymphocyte series, for example, NS0 myeloma cell and SP2 cell, COS cell and from the cells of transgenic animal, for example transgene mammal.For example, described cell is the Mammals epithelial cell.

Except the nucleotide sequence of the various immunoglobulin domains of encoding, other sequence of recombinant expression vector portability is such as regulate and control sequence (for example replication orgin) and the selectable marker gene that carrier duplicates in host cell.Described selectable marker gene promotes the selection to host cell, and described host cell is the cell (as seen, for example United States Patent (USP) 4,399,216,4,634,665 and 5,179,017) that has imported described carrier.For example, common described selectable marker gene shows the resistance to medicine, for example G418, Totomycin or methotrexate, and it is applied on the host cell that described carrier imports.Preferred selectable marker gene comprises Tetrahydrofolate dehydrogenase (DHFR) gene (dhfr that is used for methotrexate selection/amplification ^-Host cell) and neo gene (be used for G418 select).

In the recombinant expressed typical systems of antibody of the present invention or its antigen-binding portion thereof, by the transfection of calcium phosphate mediation, with the expression vector importing dhfr of encoding antibody heavy chain and light chain of antibody ^-Chinese hamster ovary celI.In recombinant expression vector, heavy chain of antibody and light chain gene (for example have been operably connected the enhancers/promoters controlling element, derive from SV40, CMV, adenovirus etc. are such as cmv enhancer/AdMLP promoter regulation element or SV40 enhanser/AdMLP promoter regulation element) high level that drives described gene transcribes.Described recombinant expression vector also carries the DHFR gene, and it allow to use methotrexate selection/amplification Chinese hamster ovary celI of described carrier of having selected transfection.Cultivate selected transformed host cell with permission heavy chain of antibody and light chain expression, and from substratum, reclaim complete antibody.Standard molecular biological technique is used for preparing recombinant expression vector, transfection host cell, selection transformant, cultivates host cell and reclaims antibody from substratum.For example, some antibody can use albumin A or Protein G to separate by affinity chromatography.

For the antibody that comprises the Fc structural domain, the synthetic Fc district of antibody producing optimum system choosing is by glycosylated antibody.For example the Fc structural domain of IgG molecule on No. 297 l-asparagines of CH2 structural domain by glycosylation.This l-asparagine is the site that two feeler type oligosaccharides are modified.Prove that this glycosylation is effector function essential (Burton and Woof, 1992, the Adv.Immunol.51:l-84 of Fc γ acceptor and C1Q mediation; Jefferis etc., 1998, Immunol.Rev.163:59-76).In a preferred embodiment, the Fc structural domain produces in mammalian expression system, and described system suitably makes the residue glycosylation corresponding to No. 297 l-asparagines.Described Fc structural domain is modified after also can comprising other eukaryotic translation.

In case recombinant expressed generation antibody molecule, can carry out purifying by the method antagonist molecule of any purifying immunoglobulin molecules known in the art, for example, by chromatogram (for example, ion-exchange, affine particularly after albumin A by with the affine and Size Exclusion Chromatograph SEC of specific antigen), the standard technique of centrifugal, solvability difference or any other protein purification.In addition, described antibody or its fragment can merge to make things convenient for purifying with allogeneic polypeptide sequence known in the art.

Gene therapy technology

Gene therapy refers to by use the treatment that nucleic acid expression or effable is implemented to object.Can use this area to can be used for any method of gene therapy according to the present invention.Typical method is as described below.

In a specific embodiments, by gene therapy methods, use one or more antisense oligonucleotides of the one or more biomarkers of the present invention, to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In other embodiments, pass through gene therapy methods, use the one or more nucleic acid molecule that comprise one or more antibody encoding nucleic acids, preventing, to treat, to control or to alleviate bladder cancer or its symptom, described antibodies specific is in conjunction with one or more proteins of one or more marks of the present invention.In other embodiments, pass through gene therapy methods, use one or more proteins of comprising the one or more biomarkers of the present invention or one or more nucleic acid molecule of its analogue, derivative or segmental coding nucleic acid, to prevent, to treat, to control or to alleviate bladder cancer or its symptom.In other embodiments, pass through gene therapy methods, use the one or more nucleic acid molecule that comprise the negative peptide coding nucleic acid of one or more dominance, to prevent, to treat, to control or to alleviate bladder cancer or its symptom, the negative polypeptide of dominance of one or more proteins that the negative polypeptide of described dominance is the one or more biomarkers of the present invention.

For the general summary of gene therapy method, visible Goldspiel etc., 1993, ClinicalPharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev.Pharmacol.Toxicol.32:573-596; Mulligan, 1993, Science260:926-932; With Morgan and Anderson, 1993, Ann.Rev.Biochem.62:191-217; May, 1993, TIBTECH 11 (5): 155-215). (eds.) such as the method Ausubel as described below of the recombinant DNA technology that available is generally known in the art, 1993, CurrentProtocols in Molecular Biology, John Wiley﹠amp; Sons, NY; And Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In one aspect, composition of the present invention comprises the nucleotide sequence of one or more antibody of encoding, described antibodies specific is in conjunction with one or more proteins of the one or more biomarkers of the present invention, and described nucleotide sequence is a part of expressing the expression vector of one or more antibody in suitable host.Especially, such nucleotide sequence has the promotor that can be operationally connected to antibody, and described promotor is derivable or composing type, and, preferably tissue-specific.

In one aspect of the method, composition of the present invention comprises the nucleotide sequence of the negative polypeptide of coding dominance, the negative polypeptide of dominance of one or more proteins that the negative polypeptide of described dominance is the one or more biomarkers of the present invention, described nucleotide sequence is a part of expressing the negative polypeptide expression carrier of dominance in suitable host.Especially, such nucleotide sequence has the promotor that can be operationally connected to the negative polypeptide of dominance, and described promotor is derivable or composing type, and, randomly be tissue-specific.In another embodiment, nucleic acid molecule, wherein the negative encoding sequence of dominance and any other required sequence flank join with the zone of impelling required site generation homologous recombination in genome, therefore intrachromosomal expression (the Koller and Smithies of the negative nucleic acid of dominance is provided, 1989, Proc.Natl.Acad.Sci.USA 86:8932-8935; Zijlstra etc., 1989, Nature 342:435-438).

Send described nucleic acid and enter the patient and directly send, the patient directly touches nucleic acid or carries the carrier of nucleic acid in this case, perhaps sends indirectly, in this case, at first uses described nucleic acid at the vitro conversion cell, then cell is implanted the patient.Known these two kinds of methods are respectively in the body or stripped gene therapy.

In a specific embodiments, use in the direct body of described nucleotide sequence, it is expressed and produces coded product in vivo.This can finish by any method in the several different methods known in the art, for example, by it being made up as the part of suitable nucleic acid expression vector and using it to make that they become intracellular, for example by using defective or attenuation retrovirus or other virus vector (to see United States Patent (USP) 4,980,286), perhaps by the direct injection naked DNA, perhaps by using microparticle bombardment (for example, particle gun; Biolistic, Dupont), perhaps, be encapsulated in liposome, particulate or the micro-capsule, perhaps knownly enter nuclear peptide and use by they are connected to by lipid or cell surface receptor or quilt that transfection reagent wraps, use and (see by it being connected to the part that is subjected to the receptor mediated endocytosis effect, for example, Wu and Wu, 1987, J.Biol.Chem.262:4429-4432) (it is used for the cell type that targeting specific is expressed described acceptor), or the like.In another embodiment, nucleic acid-ligand complex can be formed, and wherein said part comprises the fusion viral peptide with the endosome that breaks, and makes described nucleic acid avoid the lysosome degraded.In another embodiment, by being targeted to specific receptors, but in the described nucleic acid body targeted cells specificity take in and express (see, for example, 92/06180,1992 on April 16, (Wu etc.) of international open WO; 92/22635,1992 on December 23, (Wilson etc.) of WO; WO92/20316, November 26 (Findeis etc.) in 1992; 93/14188,1993 on July 22, (Clarke etc.) of WO, 93/20221,1993 on October 14, (Young) of WO).Perhaps, described nucleic acid can to import in cell and to be integrated into host cell DNA (Koller and Smithies, 1989, Proc.Natl.Acad.Sci.USA 86:8932-8935 in order expressing by the homologous recombination merging; Zijlstra etc., 1989, Nature 342:435-438).

For example, can use retrovirus.These retroviral vectors have been deleted the packaging virus genome and have been incorporated into the unwanted retroviral sequence of host DNA through modifying.Coding is used for the antibody interested of gene therapy or proteins of interest matter or its segmental nucleotide sequence and is cloned into one or more carriers, and it makes gene delivery enter the patient.Be found in Boesen etc. about the retrovirus more detailed description, 1994, Biotherapy 6:291-302, it has been described and has been applied to the retroviral vector of mdr1 gene delivery in the hemopoietic stem cell be its objective is to make stem cell more can resist chemotherapy.Other reference is for example understood the application of retrovirus in gene therapy: Clowes etc., 1994, J.Clin.Invest 93:644-651; Kiem etc., 1994, Blood83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy4:129-141; With Grossman and Wilson, 1993, Curr.Opin.in Genetics andDevel.3:110-114.

Adenovirus is the other virus vector that is used for gene therapy.Adenovirus is the attractive especially carrier that is used for delivery of gene to the respiratory epithelium cell.Adenovirus naturally infect respiratory epithelium cell causes minor ailment.Other target based on the delivery system of adenovirus has liver, central nervous system, endotheliocyte and muscle.Adenovirus has the advantage that can infect Unseparated Cell.Kozarsky andWilson, 1993, Current Opinion in Genetics and Development 3:499-503 has provided the summary based on the gene therapy of adenovirus.Bout etc., 1994, Human Gene Therapy5:3-10 has proved that adenovirus carrier is transmitting the application of gene to rhesus monkey respiratory epithelium cell.Adenovirus is used for other example of gene therapy can be at Rosenfeld etc., 1991, Science252:431-434; Rosenfeld etc., 1992, Cell 68:143-155; Mastrangeli etc., 1993, J.Clin.Invest.91:225-234; The open WO94/12649 of PCT; And Wang, etc., 1995, find among the GeneTherapy 2:775-783.In a preferred embodiment, use adenovirus carrier.

Adeno associated virus (AAV) also has been intended for use in gene therapy (Walsh etc., 1993, Proc.Soc.Exp.Biol.Med.204:289-300; United States Patent (USP) 5,436,146).

Another method of gene therapy comprises that with the cell of transgenosis in the tissue culture it is by for example method of electroporation, liposome transfection, calcium phosphate mediation transfection or virus transfection.Usually, transfer method comprises selective marker is transferred to described cell.Then select these cells to separate the cell that has had and expressed institute's metastatic gene.Give the patient with these cell deliveries then.

In this embodiment, described nucleic acid is imported in the cell, use the gained reconstitution cell then in the body.Such importing can be implemented by any known method in this area, and it includes but not limited to transfection, electroporation, microinjection, the virus that contains described nucleotide sequence or phage vector transfection, cytogamy, the transfer of karyomit(e) mediated gene, the transfer of minicell mediated gene, protoplastis fusion etc.Be used for importing foreign gene to the multiple technologies of cell be known in the art (see, for example, Loefflerand Behr, 1993, Meth.Enzymol.217:599-618; Cohen etc., 1993, Meth.Enzymol.217:618-644; Cline, 1985, Pharmac.Ther.29:69-92) and can use according to the present invention, as long as the growth and the physiological function of recipient cell necessity are not destroyed.Described technology should provide described nucleic acid stability to transfer to described cell, makes that described nucleic acid can be by cell expressing, and preferably can heredity and can be expressed by progeny cell.

The gained reconstitution cell can be delivered to the patient by multiple means known in the art.Reorganization hemocyte (for example, hemopoietic stem cell or progenitor cell) and/or the preferred intravenously of chondrocyte are used.Estimate to use the amount of cell to depend on required effect, patient's states etc., and can be determined by those skilled in the art.

The cell that can import nucleic acid for the gene therapy purpose comprise any needs, the available cell type, and include but not limited to epithelial cell, endotheliocyte, keratinocyte, chondrocyte, inoblast, myocyte, liver cell, hemocyte (for example T lymphocyte, bone-marrow-derived lymphocyte, monocyte, scavenger cell, neutrophilic granulocyte, eosinophilic granulocyte, megalokaryocyte, granulosa cell), multiple stem cell or progenitor cell (particularly hemopoietic stem cell or progenitor cell for example obtain from marrow, Cord blood, peripheral blood, fetus liver) etc.

In a preferred embodiment, the cell that is used for gene therapy is that the patient originates from body.

The embodiment that is used for gene therapy at a reconstitution cell, antibody interested or the proteins of interest matter of encoding or its segmental nucleotide sequence are imported into cell, make them to be expressed, follow described reconstitution cell and used in the body to reach result of treatment by the offspring of cell or cell.In a specific embodiments, use stem cell or progenitor cell.According to this embodiment of the present invention, any can separated and external stem cell of keeping and/or progenitor cell can be employed (see, for example, international open WO94/08598, on April 28th, 1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth.Cell Bio.21A:229; With Pittelkow and Scott, 1986, Mayo Clinic Proc.61:771).

It can be composing type, derivable or tissue-specific can be used for controlling coding antibody interested, proteins of interest matter or its segmental nucleotide sequence expression promoter.Limiting examples comprises SV40 early promoter district (Bernoist and Chambon, 1981, Nature 290:304-310), described promotor contains 3 ' the long terminal repetition district (Yamamoto of Rous sarcoma virus, et al., 1980, Cell 22:787-797), bleb thymidine kinase promotor (Wagner et al., 1981, Proc.Natl.Acad.Sci.USA 78:1441-1445), metallothionein gene regulating and controlling sequence (Brinster etal., 1982, Nature 296:39-42); Prokaryotic expression carrier is β-Nei Xiananmei promotor (Villa-Kamaroff et al. for example, 1978, Proc.Natl.Acad.Sci.USA 75:3727-3731), or tac promotor (DeBoer et al., 1983, Proc.Natl.Acad.Sci.USA 80:21-25), also visible " Useful proteins from recombinant bacteria " in Scientific American, 1980,242:74-94; Plant expression vector comprises rouge alkali synthetase promoter district (Herrera-Estrella et al., Nature 303:209-213) or cauliflower mosaic virus 35S RNA promotor (Gardner et al., 1981, Nucl.Acids Res.9:2871), promotor (Herrera-Estrella et al. with the photosynthetic enzyme diphosphoribulose carboxylase, 1984, Nature 310:115-120); Promoter element from yeast or other fungi, Gal4 promotor for example, ADC (ethanol dehydrogenase) promotor, PGK (phosphoglycerokinase) promotor, alkaline phosphatase promoter and following animal transcription regulatory region, it shows tissue specificity and has been used for transgenic animal: elastoser I gene control region, it has activity (Swift et al., 1984, Cell38:639-646 in pancreatic acinar cell; Ornitz et al., 1986, Cold Spring Harbor Symp.Quant.Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); The insulin gene control region wherein has activity (Hanahan, 1985, Nature 315:115-122) in the pancreatic beta cell, the immunoglobulin gene control region, and it has activity (Grosschedl et al., 1984, Cell38:647-658 in lymphocyte; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, MoI.Cell.Biol.7:1436-1444), the mouse mammary tumour virus control region, it has activity (Leder et al. in testis, mammary gland, lymph and mastocyte, 1986, Cell 45:485-495), the albumin gene control region, it has activity (Pinkert et al. in liver, 1987, Genes and Devel.1:268-276), the a-fetoprotein gene control region, it has activity (Krumlauf et al. in liver, 1985, MoI.Cell.Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58; The alpha1-antitrypsin gene control region, wherein liver has activity (Kelsey et al., 1987, Genesand Devel.1:161-171), the beta globin genes control region, it has activity (Mogram et al. at medullary cell, 1985, Nature 315:338-340; Kollias et al., 1986, Cell46:89-94; Myelin basic protein control region, its oligodendrocyte in brain have activity (Readhead et al., 1987, Cell 48:703-712); Myosin light chain-2 gene control region, it has activity (Sani, 1985 in skeletal muscle, Nature 314:283-286) and the gonadotropin releasing hormone gene control region, it has activity (Mason et al. at hypothalamus, 1986, Science234:1372-1378).

In a specific embodiments, for the gene therapy purpose, nucleic acid to be imported comprises inducible promoters, its exercisable coding region that is connected to, make that described expression of nucleic acids is controlled, whether it controls expression by controlling the existence of suitably transcribing inductor.

5.20 medicinal compositions

The bioactive compounds or its medicinal acceptable salt that use the inventive method to identify can be administered to the patient who suffers from bladder cancer, and preferred mammal is more preferably human.In a specific embodiments, compound or its medicinal acceptable salt can be administered to suffers from bladder cancer and/or the patient of early stage bladder cancer in late period, and preferred mammal is more preferably human.In another embodiment, the preventive measures that compound or its medicinal acceptable salt can be used as bladder cancer are administered to the patient, and preferred mammal is more preferably human.According to these embodiments, described patient can be children, grow up or old, and wherein " children " are the object of age between 24 months to 18 years old, " growing up " be the age at the object more than 18 years old, and " old age " be the object of age at over-65s.

When being administered to the patient, described compound or its medicinal acceptable salt be preferably as the component applied of composition, and described composition is optional comprises the medicinal carrier of accepting.But described composition dosage forms for oral administration, or by other any approach easily, for example, by infusion (infusion) or inject (bolus injection) fast, by absorbing, and can use jointly with another kind of biologically active agent through epithelium or through mucocutaneous (for example oral mucosa, rectum and intestinal mucosa etc.).Use can be general or partial.Multiple delivery system is known, for example, be encapsulated in liposome, particulate, micro-capsule, the capsule etc., and described system can be used for using described compound and its medicinal acceptable salt.

Application process includes but not limited in intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, the nose, in the epidural, per os, hypogloeeis, nose, in the brain, intravaginal, through skin, per rectum, by sucking or partial (particularly ear, nose, eye or skin).The pattern of using is left the medical practitioner for and is judged.In most of the cases, use and to cause compound or its medicinal acceptable salt to be discharged in the blood.

In a specific embodiments, the described compound of topical application or its medicinal acceptable salt can be expected.For example, but not as restriction, this can be by the local infusion in the surgical procedure, topical application (for example, combine with postoperative wound dressing), by injection, pass through conduit, pass through suppository, perhaps finish by implantation, described implantation is porous material, non-porous material or gel-like material, and it comprises film (for example sialastic film) or fiber.

In certain embodiments, can expect that import described compound or its medicinal acceptable salt to central nervous system, described approach comprises (intrathecal) and epidural injection in Intraventricular, the sheath by any suitable approach.Intracerebral ventricle injection can be implemented by intraventricular catheter, for example links to each other with bank (reservoir) (such as the Ommaya bank).

Also can use pulmonary administration, for example by sucker or atomizer, and the prescription of use smoke substance, perhaps pour into by fluorocarbon or synthetic Curosurf.In certain embodiments, described compound and its medicinal acceptable salt can be made into suppository, and it uses traditional tackiness agent and carrier (for example triglyceride level).

In another embodiment, described compound and its medicinal acceptable salt can be sent in bank, particularly liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, the same., pp.317-327; Usually can be referring to above).

In another embodiment, described compound and its medicinal acceptable salt can be sent (see, for example, Goodson, in Medical Applications of Controlled Release, as above, vol.2, pp.115-138 (1984)) in controlled release system.Can use summary Langer, 1990, other controlled release system of being discussed among the Science249:1527-1533.In one embodiment, can use pump (to see Langer, as above; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl.J.Med.321:574).In another embodiment, can use polymer materials (to see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol.Sci.Rev.Macromol.Chem.23:61; Also visible Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol.25:351; Howard et al., 1989, J.Neurosurg.71:105).In another embodiment, controlled release system can be placed near the target RNA of compound or its medicinal acceptable salt, therefore only needs the sub-fraction of systemic doses to get final product.

Compound as herein described can be impregnated in the medicinal compositions that is suitable for using.Such composition comprises described active compound and the medicinal carrier of accepting usually.Term used herein " the medicinal carrier of the accepting " meaning comprise any He all solvents, dispersion medium, dressing, antibacterium and anti-mycotic agent, etc. blend delay absorption agent etc., it is compatible with medicament administration.The application that such medium and preparation are used for active medicinal matter is well known in the art.Except any traditional and inconsistent medium of described active compound or preparation, can consider that all it is applied to composition.Additional active compound also can mix described composition.

The present invention includes the method that preparation is used to regulate polypeptide of interest or expression of nucleic acids or active medicinal compositions.Such method comprises medicinal carrier and the interested polypeptide of adjusting or expression of nucleic acids or the active medicament accepted of preparation.Such composition also can comprise some other active substance.Therefore, the present invention also comprises the method for preparing medicinal compositions, and it is medicinally accepted carrier and regulate polypeptide of interest or other active compound of expression of nucleic acids or active medicament and one or more by preparing.

The route of administration that medicinal compositions of the present invention is formulated into its expection adapts.The example of route of administration comprises parenteral, for example, intravenously, cortex are interior, subcutaneous, per os (for example sucking), (part) of transdermal, saturating mucous membrane and rectal administration.It is preferred that intravenously is used.The solution or the suspension that are used for parenteral, intracutaneous or subcutaneous administration can comprise following composition: sterile diluent (for example water for injection, salts solution, expressed oil, polyoxyethylene glycol, glycerine, propylene glycol or other synthetic); Antiseptic-germicide (for example phenylcarbinol or methyl p-hydroxybenzoate); Antioxidant (for example xitix or sodium bisulfite); Sequestrant (for example ethylenediamine tetraacetic acid (EDTA)); Buffer reagent (for example acetate, Citrate trianion or phosphoric acid salt) and the material (for example sodium-chlor or glucose) that is used to change osmotic pressure.Use acid or alkali to regulate pH, for example hydrochloric acid or sodium hydroxide.The parenteral prepared product can be encapsulated in ampoule, disposable syringe or the multiple dose vials that glass or plastics make.

The sterilized powder that the medicinal compositions that is suitable for injecting purposes comprises aseptic aqueous solution (water-soluble) or dispersion system and is used for preparing immediately aseptic injectable solution or dispersion system.When being used for intravenously and using, suitable carriers comprises physiological saline, bacteriostatic water, Cremophor EL ^TM(BASF; Parsippany, NJ) or phosphate buffered saline buffer (PBS).In all cases, described composition must be aseptic and should be fluid that its fluid degree is to make to make things convenient for syringe to use.It must be stable under manufacturing and preservation condition, and must prevent the pollution of microorganism (for example bacterium and fungi).Described carrier can be solvent or dispersion medium, and it comprises, for example, and water, ethanol, polyol (for example glycerine, propylene glycol and liquid macrogol etc.) and suitable mixture thereof.Can keep suitable flowability, for example, by using dressing (for example Yelkin TTS), by under the situation of dispersion system, keeping required granular size, and by using tensio-active agent.Prevent that action of microorganisms from can finish by multiple antibacterium and anti-mycotic agent (for example P-hydroxybenzoic acid class, butylene-chlorohydrin, phenol, xitix, Thiomersalate etc.).In many cases, it will preferably comprise isotonic agent (isotonic agent) in composition, for example, and carbohydrate, polyalcohols (for example N.F,USP MANNITOL, sorbyl alcohol), sodium-chlor.Can bring the prolongation of composition for injection to absorb by in composition, comprising the material (for example aluminum monostearate) that postpones to absorb.

Aseptic injectable solution can prepare by following steps: the active compound (for example polypeptide or antibody) of aequum is added in the suitable solvent, and described solvent comprises a kind of composition or the composition combination of above enumerating, subsequently filtration sterilization as required.Usually, prepare dispersion system in the sterile carrier by active compound is joined, described sterile carrier contains basic dispersion medium and required other above cited composition.Under the situation of the sterilized powder that is used to prepare aseptic injectable solution, the preferred preparation method is vacuum-drying and lyophilize, the activeconstituents of filtration sterilization solution and the powder of any other required composition before it obtains coming from.

The composition of per os comprises inert diluent or edible carrier usually.They can be packaged in gelatine capsule or be pressed into tablet.For the purpose of per os therapeutic administration, described active compound can with mixed with excipients, and use with tablet, lozenge or capsular form.The composition of per os also can use fluid carrier to prepare, and it is used to gargle, and wherein the described compound in the fluid carrier is dosage forms for oral administration, gargles, and spues then or swallows.

Medicinal compatible adhesive, and/or the part that subsidiary material can be used as described composition is included.Tablet, pill, capsule, lozenge etc. can contain following any composition, perhaps the compound of similar performance: tackiness agent (for example Microcrystalline Cellulose, tragacanth gum or gelatin); Vehicle (for example starch or lactose), disintegrating agent (for example alginic acid, Primogel or W-Gum); Lubricant (for example Magnesium Stearate or Sterotes); Glidant (for example colloid silica); Sweeting agent (for example sucrose or asccharin); Perhaps perfume compound (for example peppermint, wintergreen oil or lemon flavour).

For using by suction, described compound is sent with the form of aerosol spray, and it is from pressurized vessel or divider, and it contains suitable propellent, for example gas (such as carbonic acid gas), or atomizer.

General is used also can be by mode saturating mucous membrane or transdermal.Use for saturating mucous membrane or transdermal, in prescription, use the permeate agent that is suitable for carrier to be penetrated.Such permeate agent is normally known in the art, comprises, for example, and for saturating mucosal administration, stain remover, cholate and fusidic acid derivatives.Passing mucosal administration can finish by using nose spraying or suppository.Use for transdermal, described active compound is formulated into ointment (ointment), ointment (salve), gel or creme as generally known in the art.

Described compound also can be formulated into suppository form (for example, using traditional suppository base (for example theobroma oil or other glyceryl ester)) or be used for the enema forms that rectum is sent.

In one embodiment, described active compound prepares with carrier, and described carrier will protect described compound to avoid the quick discharge of health, controlled release formulation for example, and it comprises implants and the micro-capsule delivery system.Can use biodegradable, bioavailable polymer, for example ethylene vinyl acetate, poly-anhydrides, polyglycolic acid, collagen, poe class and poly(lactic acid).The preparation method of prescription like this will be tangible for a person skilled in the art.Described material also can be from Alza Corporation and Nova Pharmaceuticals, and Inc. buys.Liposome turbid liquor (liposome and the antiviral antigenic monoclonal antibody that comprise the infected cell of target) also can be used as the medicinal carrier of accepting.These can prepare by method known to those skilled in the art, as United States Patent (USP) 4,522, described in 811.

Preparation is particularly advantageous with per os or the parenteral composition that dosage unit form exists, and it conveniently uses and have the homogeneity of dosage.Dosage unit form used herein refers to physically separated unit, and it is suitable for the unitary dose of the object to be treated of opposing; Each unit contains the active compound that can produce expected effect as calculated of predetermined amount, and it has united required pharmaceutical carrier.The specification of dosage unit form of the present invention be according to and directly depend on the specific characteristic of described active compound and the specific therapeutical that will reach, and in this area to the inherent limitations of this class active compound of being mixed for individual treatment.

For antibody, preferred dose is that the 0.1mg/kg body weight is to 100mg/kg body weight (more preferably 0.1 to 20mg/kg, 0.1 arrive 10mg/kg, perhaps 0.1 arrive 1.0mg/kg).If described antibody works in brain, 50mg/kg is suitable to the dosage of 100mg/kg usually.Usually, groups of people's antibody and whole person's antibody have the transformation period long with respect to other antibody in human body.Therefore, lower dosage and lower frequency of administration often are possible.Modifying (for example fatization) can be used for stabilization of antibodies and increases taking in and tissue penetration (for example, entering in the brain).Cruikshank etc. (1997, J.Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193) have described the method for fat antibody.

In a specific embodiments, the significant quantity of protein or polypeptide (being effective dose) scope is about 0.001 to the 30mg/kg body weight, preferred about 0.01 to the 25mg/kg body weight, more preferably from about 0.1 to the 20mg/kg body weight, in addition more preferably from about 0.1 to 1.0mg/kg body weight, 1 to 10mg/kg body weight, 2 to 9mg/kg body weight, 3 to 8mg/kg body weight, 4 to 7mg/kg body weight or 5 to the 6mg/kg body weight.

The technician will understand some factor may influence the necessary dosage of effective treatment target, and it includes but not limited to the severity of disease or illness, previous treatment, holistic health and/or object age and other disease that exists.In addition, protein, polypeptide or the antibody of use treatment significant quantity can comprise single therapy to the treatment of object, and is perhaps preferred, can comprise a series of treatments.

Except those compounds as described above, the present invention comprises adjusting nucleic acid interested or polypeptide expression or active micromolecular application.Micromolecular limiting examples comprises peptide, intends peptide, amino acid, amino acid analogue, polynucleotide, polymerized nucleoside acid-like substance, Nucleotide, nucleotide analog, has less than about 10, the 000g/mol molecular weight, have less than about 5, the 000g/mol molecular weight, have, the 000g/mol molecular weight, have the organic or inorganic compound (promptly comprising different organic (heteroorganic) and organometallic compound) less than about 500g/mol molecular weight and salt, ester and other medicinal form of accepting of these compounds less than about 1.

Can understand, the suitable dose of small-molecule substance depends on many factors, and it is in gengral practitioner, animal doctor or researchist's ken.Micromolecular dosage is with basis, for example, the feature of object or sample to be treated, size and situation and change, to change according to the approach of applying said compositions in addition, if feasible, and will expect that small molecules changes to nucleic acid of the present invention or polypeptide role according to the doctor.Typical doses comprises the small molecules (for example, every kilogram to about 500 milligrams every kilogram of about 1 microgram, every kilogram to about 5 milligrams every kilogram of about 100 microgram, every kilogram of perhaps about 1 microgram is to every kilogram of about 50 microgram) of every kilogram of object or sample body weight milligram or microgram amount.In addition, can understand micromolecular suitable dose and depend on that small molecules is for waiting to regulate expression or active effectiveness.Such suitable dose can use mensuration described herein to determine.When object (for example, the people) is used these micromolecular one or more, to regulate polypeptide of the present invention or expression of nucleic acids or when active, doctor, animal doctor or researchist can, for example, at first provide the prescription of relative low dosage, then increase dosage until obtaining suitable reaction.In addition, can understand, given dose level for any particular animals object will depend on multiple factor, it comprises the activity of the specific compound that uses, age, body weight, holistic health, sex and the diet of described object, time of application, route of administration, discharge rate, any drug regimen, and expression to be regulated or level of activity.

Described medicinal compositions can be included in container, packing or the divider with operation instruction.

5.21 test kit

The invention provides the test kit of the expression of the protein of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 of being used to measure biomarker of the present invention, all or any combination and RNA product.Such test kit comprises necessary material of expression and the reagent of measuring these protein and RNA product.In a specific embodiments, described test kit can comprise one or more employed other reagent in several different methods in addition, and for example: (1) is used for stablizing and/or being used to produce from the reagent (2) of blood or tissue purifying RNA the primer of test nucleic acid; (3) dNTP and/or rNTP (be pre-mixed or separate), optional dNTP and/or rNTP (plain dNTP mark or that have Cy3 or Cy5 label of biological example) with one or more specific markers; (4) synthesize back labelled reagent, for example the chemically reactive derivative of fluorescence dye; (5) enzyme, for example reversed transcriptive enzyme, archaeal dna polymerase etc.; (6) numerous buffers, for example, reaction, hybridization and washing buffer; (7) mark the purified reagent and the component of probe, centrifugal post etc. for example; And (8) protein purification reagent; (9) signal produces and detection reagent, for example streptavidin-alkaline phosphatase conjugate, chemiluminescence or chemical luminous substrate etc.In a specific embodiments, the separation that described test kit comprises mark in advance from sample (for example, blood or chondrocyte or synovia) Quality Control reference protein and or RNA, it is with comparing.

In some embodiments, described test kit is RT-PCR or qRT-PCR test kit.In some other embodiment, described test kit is nucleic acid array and protein array.Such test kit according to the present invention will comprise array and the Packaging Method thereof with related protein of the present invention or nucleic acid member at least.As an alternative, described protein of the present invention or nucleic acid member can be packaged on the array in advance.

In some embodiments, described test kit is a quantitative RT-PCR kit.In one embodiment, quantitative RT-PCR kit comprises: each primer of biomarker combination of the present invention (a) is used for increasing; (b) buffer reagent and enzyme (it comprises reversed transcriptive enzyme); (c) one or more heat-stabilised poly synthase; (d)

Green.In another embodiment, test kit of the present invention also comprises (a) with reference to contrasting RNA and (b) mixing contrast RNA.

The invention provides and be used for the individual test kit of suffering from bladder cancer and/or early stage bladder cancer of (a) diagnosis.For example, in particular of the present invention, test kit comprises the upstream and downstream primer, wherein forward and reverse primer are designed to the mRNA corresponding to each biomarker of identifying according to the present invention of all species of quantitative expression, and described biomarker is used for determining whether individuality suffers from bladder cancer and/or early stage bladder cancer.In certain embodiments, one of described at least primer is to be designed to stride across exon connect.

The invention provides be used to detect, the test kit of diagnosis, monitor and predict bladder cancer, it is based in sample, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 of biomarker of the present invention, all or the protein of any combination or the expression of RNA product.In certain embodiments, such test kit does not comprise and is used for measuring biomarker of the present invention and shows the protein of the biomarker relevant with bladder cancer or material and the reagent that the RNA product is expressed by prior art.In other embodiments, such test kit comprises and is used for measuring material and the reagent that biomarker of the present invention has shown the biomarker relevant with bladder cancer by prior art and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or more polygenic protein or RNA product are expressed except that biomarker of the present invention.

The invention provides the test kit that is used for one or more treatment curative effects that monitored object accepting, it is based in sample, is up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the protein of any amount of biomarker of the present invention of all or any combination or the expression of RNA product.In certain embodiments, such test kit does not comprise and is used for measuring biomarker of the present invention and shows the protein of the biomarker relevant with bladder cancer or material and the reagent that the RNA product is expressed by prior art.In other embodiments, such test kit comprises and is used for measuring biomarker of the present invention and has shown the biomarker relevant with bladder cancer by prior art and be up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or the protein of more kinds of any amount genes or material and the reagent that the RNA product is expressed except that biomarker of the present invention.

The invention provides and be used for determining whether object responds the test kit of treatment, it is based in sample, is up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the protein of any amount of biomarker of the present invention of all or any combination and the expression of RNA product.In certain embodiments, such test kit does not comprise and is used for measuring biomarker of the present invention and shows the protein of the biomarker relevant with bladder cancer or material and the reagent that the RNA product is expressed by prior art.In other embodiments, such test kit comprises and is used for measuring biomarker of the present invention and has shown the biomarker relevant with bladder cancer by prior art and be up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or the protein of more kinds of any amount genes or material and the reagent that the RNA product is expressed except that biomarker of the present invention.

The invention provides and be used for being determined at sample, be up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the test kit of the expression of the RNA product of any amount of biomarker of the present invention of all or any combination.In a specific embodiments, such test kit comprises mensuration biomarker RNA product of the present invention and expresses necessary material and reagent.For example, can produce the microarray or the RT-PCR test kit that are used for bladder cancer, it contains for measurement and is up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the necessary reagent of RNA product level and the material of any amount biomarker of the present invention of all or any combination.As an alternative, in some embodiments, described test kit can comprise and is not limited to measure the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the necessary reagent of RNA product level and the material of any amount biomarker of the present invention of all or any combination.For example, except be up to few 1 for measurement, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, outside the necessary reagent of RNA product level and material of any amount biomarker of the present invention of all or any combination, the microarray test kit can contain measures the essential reagent and the material of RNA product level of must relevant or not indicate bladder cancer with bladder cancer.In a specific embodiments, microarray or RT-PCR test kit contain to be useful on to measure and are up to few 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, the necessary reagent of RNA product level and the material of any amount of biomarker of the present invention of all or any combination, and it is the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,175,200,225,250,300,350,400,450 or more any amount of biomarker of the present invention are with the necessary reagent of RNA product level and material, the perhaps 1-10 of alia gene, 1-100,1-150,1-200,1-300,1-400,1-500,1-1000,25-100,25-200,25-300,25-400,25-500,25-1000,100-150,100-200,100-300,100-400,100-500,100-1000,500-1000 mark of the present invention is with the necessary reagent of RNA product level and the material of alia gene.

For the nucleic acid microarray test kit, described test kit generally comprises the probe that is connected solid support surface.Can use detectable label to come the described probe of mark.In a specific embodiments, described probe be the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, exon, intron, exon junction, the exon-intron junction of the RNA product of any amount biomarker of the present invention of all or any combination are specific.Described microarray test kit can comprise the explanation of implementing mensuration and method, to explain and to analyze and implement to measure resulting data.In a specific embodiments, described test kit comprises the explanation that is used for diagnosing bladder cancer.The necessary reagent of signal that described test kit produces in the time of also can comprising hybridizing reagent and/or be used for detection probe to target nucleic acid sequence.Usually, be used for the material of microarray test kit and reagent at one or more containers.Each component of described test kit is usually located at it separately in the suitable containers.

For the RT-PCR test kit, described test kit generally comprise in advance select the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the specific RNA product of any amount biomarker of the present invention of all or any combination (for example exon, intron, exon connect, exon-intron connect) are had specific primer.Described RT-PCR test kit also can comprise the enzyme that is suitable for reverse transcription and/or amplification of nucleic acid (for example polysaccharase is such as Taq) and the buffer reagent of deoxynucleotide and reverse transcription and amplification reaction mixture needs.That described RT-PCR test kit also can comprise is the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the specific probe of the RNA product of any amount biomarker of the present invention of all or any combination.Can use detectable label (for example fluorescent mark) to come mark or the described probe of mark not.Each composition of RT-PCR test kit generally is arranged in its oneself suitable vessel.Therefore, these test kits generally comprise and are suitable for every kind of reagent, enzyme, primer and probe unique container separately.In addition, described RT-PCR test kit can comprise the explanation that is used to implement to measure with method, explains and analyzes and implement to measure resulting data being used for.In a specific embodiments, described test kit contains the explanation that is useful on diagnosing bladder cancer.

In a specific embodiments, described test kit is the real-time RT-PCR test kit.Such test kit can comprise 96 orifice plates and SYBR Green detects essential reagent and material.Described test kit can comprise reagent and material makes the β Actin muscle can be used for making result standardization (normalize).Described test kit also can comprise contrast, for example water, phosphate buffered saline(PBS) and phage MS2RNA.In addition, described test kit can comprise the explanation that is used to implement to measure with method, explains and analyzes and implement to measure resulting data being used for.In a specific embodiments, described explanation regulation, the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the RNA product level of any amount biomarker of the present invention of all or any combination should measure under two concentration, described two concentration should differ, for example, 5 times to 10 times.

For test kit based on antibody, described test kit can comprise, for example: (1) is in conjunction with the first antibody of proteins of interest matter (for example, the highest 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50, the protein of any amount biomarker of the present invention of all or any combination); With, optional, (2) in conjunction with second different antibodies of described protein or first antibody, it has connected detectable label (for example, fluorescent mark, radio isotope or enzyme).Test kit based on antibody also can comprise the pearl of implementing immunoprecipitation.Generally be arranged in its oneself suitable vessel based on each component of the test kit of antibody.Therefore, these test kits generally comprise the unique container that is suitable for every kind of antibody.In addition, described test kit based on antibody can comprise the explanation that is used to implement to measure with method, explains and analyzes and implement to measure resulting data being used for.In a specific embodiments, described test kit contains the explanation that is useful on diagnosing bladder cancer.

6. embodiment

Embodiment hereinafter is nonrestrictive, and is the representative in many aspects of the present invention and the characteristic.

Embodiment 1

Micro-array construction

According to the array of following steps structure according to one aspect of the invention.

At the same 96 hole pipes that are used for increasing, the PCR product that the ethanol sedimentation that uses the 3M sodium-acetate (pH5.2) of 4ul (1/10 volume) and 100ul (2.5 times of volumes) is cloned from the cDNA in OA cartilage cDNA library (～40ul), and spend the night-20 ℃ of preservations.Then that they are centrifugal 1 hour with 4 ℃ of the rotating speeds of 3300rpm.Wash gained precipitation, recentrifuge 30 minutes with 70% ice-cold ethanol of 50ul.Then with described precipitation dry air, and be resuspended in fully among 50% methyl-sulphoxide (DMSO) or the 20ul 3 * SSC and spend the night.Then use automatization GMS417 or 427 array instrument (Affymetrix, CA) single or double is positioned over γ aminopropyl silane (Gamma Amino Propyl Silane) (Corning CMT-GAPS or CMT-GAP2 with described sample, catalog number (Cat.No.) 40003,40004) or the slide of polylysine bag quilt (Sigma catalog number (Cat.No.) P0425).The border that DNA is ordered on the microarray uses diamond scribe to mark.The invention provides array, 10-20 wherein, 000 PCR product is put prepares array on solid support.

Came the described array of rehydration (described naming a person for a particular job expanded slightly, but can not be in contact with one another) in about one minute by slide being dipped in the warm no particle distilled water culture dish, then 3 seconds of rapid drying in 70-80 ℃ of upset well heater.Then UV-crosslinked (65mJ-is provided with and is shown as " 650 " for Stratagene, Stratalinker, and it is 650 * 100uJ) or 80 ℃ and cured two to four hours to slide with DNA.Described array places on the slide frame.Prepare empty slide chamber and pour into following solution: the succinyl oxide (Aldrich) of 3.0 grams is dissolved in the 1-Methyl-2-Pyrrolidone (it is vital adding reagent fast) of 189ml; After last a slice succinyl oxide dissolving, add 21.0ml 0.2M Sodium Tetraborate at once and mix, pour described solution into the slide chamber then.Described slide frame inserts in the slide chamber fast and uniformly, up and down the thermal agitation several seconds, makes slide not leave solution all the time, mixes 15-20 minute on rail mounted jolting device then.Then, described slide frame is put into 95 ℃ distilled water 2 minutes gently, put into 95% ethanol then 5 times.Then, by unnecessary ethanol is dropped on the paper handkerchief described slide dry air.Be kept in the room temperature slide box described array stand-by then.

Embodiment 2

Never isolation of RNA in the classification whole blood

(a) cracking blood

(Becton Dickinson, Franklin Lakes are saved to N.J.) and on ice it are handled (in 6 hours) to EDTA vacuum test tube (vacutainer tube) with ten milliliters of periphery whole blood collections.After centrifugal, blood preparation is divided into blood plasma, buffy coat and red corpuscle layer.Remove blood plasma and add hypotonic buffer liquid (1.6mM EDTA, 10mM KHCO with 3: 1 volume ratios ₃, 153mM NH ₄Cl pH7.4) is used for splitting erythrocyte.Centrifugal mixture obtains cell precipitation, according to the explanation dissolution precipitation of manufacturers and be dispersed in 1.0ml's

Reagent (InvitrogenCorp., Carlsbad, CA) and the 0.2ml chloroform in.After centrifugal, add Virahol to water, make it-20 ℃ of precipitations with 1: 1 ratio.The ensuing centrifugal RNA precipitation that obtains is resuspended in it in water, to be used for experiment.Specified as manufacturers, the quality of RNA is assessed on Agilent 2000Bioanalyzer RNA 6000Nano Chips, and the amount of RNA is determined by the absorbancy at 260nm place in the Beckman-Coulter DU640 spectrophotometer.

(b) centrifugal cracked blood

Obtain the 10ml whole blood place vacuum test tube and 4 ℃ with 2, centrifugal 5 minutes of 000rpm (800g) removes plasma layer then.Ratio with 1 part of blood of 3 parts of lysis buffers adds lysis buffer (lysis buffer (1L) 0.6g EDTA in blood sample; 1.0g KHCO2,8.2g NH4Cl transfers pH to 7.4 (using NaOH)).Biased sample also places on ice 5-10 minute to transparent.With the cracked sample 4 ℃ with 1000rpm centrifugal 10 minutes, the sucking-off supernatant.Resuspended precipitation in the 5ml lysis buffer, at 4 ℃ with 1000rpm recentrifuge 10 minutes.Use ratio homodisperse sedimentary cell and the abundant mixing of TRIzol (GIBCO/BRL) with the about 6ml TRIzol of the original blood sample of every 10ml.Sample was placed 5 minutes in room temperature.Every 1ml TRIzol uses 1.2ml chloroform extraction RNA.Sample 4 ℃ with 12,000 * g centrifugal 5 minutes, collect the upper strata.Ratio with every 1ml TRIzol 0.5ml Virahol adds Virahol to the upper strata.Sample spends the night or placed one hour at-20 ℃-20 ℃ of placements.According to the currently known methods precipitated rna, dry air RNA precipitation, resuspended precipitation in the distilled water that DEPC handles then.Also can be in 75% ethanol with the RNA sample retention, sample is stable for transportation at room temperature therein.

(c) from the serum-free whole blood

Obtain the 10ml whole blood place vacuum test tube and 4 ℃ with 2, centrifugal 5 minutes of 000rpm (800g) removes plasma layer then.Use ratio homodisperse sedimentary cell and the abundant mixing of TRIzol (GIBCO/BRL) with the about 6mlTRIzol of the original blood sample of every 10ml.Sample was placed 5 minutes in room temperature.Every 1ml TRIzol uses 1.2ml chloroform extraction RNA.Sample 4 ℃ with 12,000 * g centrifugal 5 minutes, collect the upper strata.Ratio with every 1ml TRIzol 0.5ml Virahol adds Virahol to the upper strata.Sample spends the night or placed one hour at-20 ℃-20 ℃ of placements.According to the currently known methods precipitated rna, dry air RNA precipitation, resuspended precipitation in the distilled water that DEPC handles then.Also can be in 75% ethanol with the RNA sample retention, sample is stable for transportation at room temperature therein.

(d) use PAXGENE ^TMFrom whole blood

Collect the 2.5ml whole blood, place PAXgene blood rna pipe, and according to PAXgene ^TMThe explanation of blood rna test kit scheme is handled.In brief, blood is at PAXgene ^TMAfter preserving at least 2 hours in the pipe, centrifugal described blood sample and supernatant discarded.Add buffer B R1 that 360ul provided in last sample, and sample is moved into centrifugal post, centrifugal then, wash through washing step repeatedly then, last wash-out is also preserved.

(e) use PAXGENE ^TMFrom whole blood, reduce globin subsequently

With aftertreatment as mentioned described in (d) from PAXgene ^TMIsolating RNA, optionally removing globin mRNA, its as

It is described to be entitled as " globin minimizing scheme " in the technical specification.α 1, α 2 and the specific oligonucleotide of beta globin are with oligonucleotide hybridization buffer and RNAse H (it is used for selectively targeted degraded globin mRNA) and remove post (it is used to remove globin mRNA) from the cRNA of Affymetrix and hatch.

Embodiment 3

Preparation and hybridization target nucleic acid

Prepare the fluorescent DNA probe from mRNA

The fluorescent mark target nucleic acid sample of preparation RNA is to analyze array of the present invention.

Kept 10 minutes by being heated to 70 ℃, then in cooled on ice, 1 μ g oligomerization dT primer is separated total RNA annealing that self diagnosis is the not classification whole blood of bladder cancer patients with 10 μ g, its cumulative volume is 10 μ l.The medical practitioner makes the diagnosis of patient's bladder cancer positive or negative.It after the positive colon pathological diagnosis histological examination in vitro tissue.By hatching 40 minutes described mRNA of reverse transcription of sample at 42 ℃, its volume is 25 μ l, and containing final concentration is 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl ₂, 25mM DTT, the unmarked dNTP of 25mM, the Superscript II of 400 units (200U/ μ l, Gibco BRL) and 15mM Cy3 or Cy5 (Amersham).Add the NaOH termination reaction of EDTA and the 5 μ l 1M of 2.5 μ l 500mM, hatched 10 minutes at 65 ℃.By adding TrisHCl (pH7.6) the neutralization reaction mixture of 12.5 μ l 1M.

The target nucleic acid sample of mark is by coming purifying by centrifugal in Centricon-30 microconcentrator (Amicon).

Heat the nucleic acid denaturation that made mark in 2 minutes at 100 ℃, hatched 20-30 minute at 37 ℃ then, then be placed on the nucleic acid array under 22mm * 22mm cover glass.Keeping by the little reservoir of 3 * SSC in the customization slide chamber of humidity, hybridizing 14 to 18 hours for 65 ℃.By its immersion being contained the 2X SSC of 0.1%SDS and shaking and cleaned described array in 2-5 minute, then be 1XSSC, 0.1X SSC then.At last, by centrifugal 2 minutes dry described arrays on the slide frame, described centrifugal be in Beckman GS-6 desk centrifuge on the Microplus frame centrifugal 2 minutes with 650rpm.

The total RNA of mark (5 μ g) and other be from the sample of suffering from or not suffering from the similar preparation of bladder cancer individuality, and at Affymetrix U133Plus 2.0GeneChips (Affymetrix; SantaClara CA) goes up hybridization, and hybridization is to carry out according to the explanation of manufacturers.In brief, in Affymetrix GCOS software (1.1.1 version), measure hybridization signal, wherein use and measure the factor, determine to measure the factor by average (the global trimmed mean) signal strength values to 500 of the overall truncation of regulating each array, and import to 7.2 editions (Silicon Genetics of GeneSpring; Redwood City, CA).Then with strength of signal (centered) placed in the middle to 50% of each chip, and for each independent gene, the intensity intermediate value to full sample sets placed in the middle.Each sample sets at least 80% in, have only by GCOS software and think that the gene that exists or be in the border just is used to further analysis.Use 1) non-parametric Wilcoxon-Mann-Whitney nonparameter test (P＜0.05), 2) parameter test (P＜0.05) and 3) non-supervision analytical method (unsupervisedanalysis method) (14) identifies the gene of differential expression.In non-supervision was analyzed, the filterable gene of strength of signal was used for, and at least 15% sample, selected expression level to have the gene of 2 times of variations (last or following) at least.Each is relatively implemented level cluster analysis (hierarchical cluster analysis) with the evaluation correlation analysis, it uses in each genes identified group sample room Spearman dependency as the tolerance of similarity, and it has used average barycenter association (average centroid linkage) in GeneSpring 6.0 editions.In table 1, provided the experiment gained result who relatively suffers from the individuality of bladder cancer and do not suffer from the individuality of bladder cancer.In table 4, provided the experiment gained result who relatively suffers from the individuality of early stage bladder cancer and do not suffer from the individuality of bladder cancer.The individuality of relatively suffering from bladder cancer and the experiment gained result who suffers from the individuality of carcinoma of testis or renal cell carcinoma in table 11, have been provided.Hereinafter select gene in table 10 indicator gauge 1 in institute's genes identified as bladder cancer biomarkers.These genes are useful especially, because they have confirmed that the remarkable multiple with remarkable p value changes.

Embodiment 4

Real-time quantitative RT PCR

Use real-time quantitative RT PCR (QRT-PCR) to carry out quantitatively from the gene that uses selection that Affymetrix U133Plus 2.0GeneChips identifies and that hereinafter mentioned in the table 13 in the table 1.Table 13 provides the tabulation of selected biomarker.Table 14 provides the tabulation of the product of the biomarker that table 13 mentions.The result of the real-time quantitative RT-PCR of use primer that table 15 is mentioned is summarised in expression in the table 16.Use Qiagen's

Green test kit (production number 204143) and/or use Applied Biosystems PCR test kit (catalog number (Cat.No.) 4334973) are finished QRT-PCR.Use Prism 7500 instruments (Applied Biosystems) to detect amplicon in real time.

At first use the High-Capacity cDNA Archive Kit (production number 4322171) of Applied Biosystems and according to use therein scheme implementation reverse transcription.

More specifically, purified RNA was hatched 10 minutes at 25 ℃ with reverse transcription damping fluid, dNTP, random primer and reversed transcriptive enzyme as mentioned before, then hatched two hours at 37 ℃, and the mixture of gained is as the initial product of quantitative PCR.

The cDNA of reverse transcription gained and the QuantiTect that is provided

Green PCRMaster Mix is hatched together, does not change magnesium ion concentration.Do not add uridylic-N-glycosylase.The upstream primer and the downstream primer that add 5 μ M gene specific of the present invention, the incubation reaction system, and use ABI PRISM 7700/ABI GeneAmp 5700/iCycler/DNA Engine Opticon to monitor according to standard scheme.Use the upstream and downstream primer of " PrimerQuest " design candidate biomarker. Http:// biotools.idtdna.com/primerquest(Integrated DNATechnologies, Coralville LA) can find instrument.Determine the Δ Ct observed value of each described gene with respect to house-keeping gene (β Actin muscle).The molten chain temperature [Tm] of thermal dissociation, and the formation of confirming specific pcr amplification and confirm not have fully primer dimer in each reaction is provided in the check of carrying out on the sepharose.Analyze following calculating of change of Ct value: Δ Ct=Ct (target gene)-Ct (house-keeping gene) between each gene and β Actin muscle house-keeping gene for each target gene.

QRT PCR also may be implemented on the RNA product of disclosed all the other biomarkers in the table 1, to the product that table 3 is mentioned, it uses, for example Qiagen

Green test kit (production number 204143).

Can use a step (reverse transcription and PCR merge) or two steps (at first reverse transcription, PCR then).Under the situation of two step schemes, at first use the High-Capacity cDNAArchive Kit (production number 4322171) of Applied Biosystems to implement reverse transcription, it is according to wherein used scheme.

More specifically, purified RNA was hatched 10 minutes at 25 ℃ with reverse transcription damping fluid, dNTP, random primer and reversed transcriptive enzyme as mentioned before, then hatched two hours at 37 ℃, and the mixture of gained is as the initial product of quantitative PCR.

The cDNA of reverse transcription gained and the QuantiTect that is provided

Green PCRMaster Mix is hatched together, does not change magnesium ion concentration.Uridylic-N-glycosylase is chosen wantonly.The forward primer and the reverse primer that add 5 μ M gene specific of the present invention, the incubation reaction system, and use ABI PRISM 7700/ABI GeneAmp 5700/iCycler/DNA Engine Opticon to monitor according to standard scheme.

Table 13

The selection of biomarker in the table 1.Form has provided Hugo gene name (gene symbol), Entrez gene I (locus link (locus link) in the past) and gene explanation.

Gene symbol	Gene I	Note
			??CHD2	??1106	Human chromatin structural domain uncoiling enzyme dna conjugated protein 2 (CHD2), mRNA
??CSPG6	??9126	People's chondroitin sulfate proteoglycan 6 (bamacan) (CSPG6), mRNA
			??CTSD	??1509	Human cathepsin D (lysosome aspartate protease) (CTSD), mRNA
??FREB	??84824	The people Fc receptor homolog thing (FREB) of expressing in the B cell, mRNA

??GAS7	??8522	People's pause specific proteins 7 (GAS7) of growing is transcribed variant b, mRNA
			??GAS7	??8522	People's pause specific proteins 7 (GAS7) of growing is transcribed variant c, mRNA
??HIST1H1C	??3006	Human histone 1H1c (HIST1H1C), mRNA
			??IGFBP7	??3490	White 7 (IGFBP7) of human insulin-like growth factor binding protein, mRNA
??IRAK3	??11213	Human interleukin-1 receptor-associated kinase 3 (IRAK3), mRNA
			??IREB2	??3658	People's iron response element binding protein 2 (IREB2), mRNA
??MLH3	??27030	People mutL homologue 3 (intestinal bacteria) (MLH3), mRNA
			??MTHFS	??10588	The people 5,10-anhydroleucovorin synthetic enzyme (5-Formyltetrahydrofolate cyclo-ligase) (MTHFS), mRNA
??NELL2	??4753	People NEL sample albumen 2 (chickens) (NELL2), mRNA
			??PLDN	??26258	People's luetin homologue (mouse) (PLDN), mRNA
??RPS24	??6229	Human rebosomal protein S24 (RPS24) transcribes variant 1, mRNA
			??RPS24	??6229	Human rebosomal protein S24 (RPS24) transcribes variant 2, mRNA
??SNTB2	??6645	People's flesh is supported conjugated protein, and β 2 (the conjugated protein A1 of dystrophin, 59kDa, basal component 2) (SNTB2), transcribes variant 1, mRNA
			??SNTB2	??6645	People's flesh is supported conjugated protein, and β 2 (the conjugated protein A1 of dystrophin, 59kDa, basal component 2) (SNTB2), transcribes variant 2, mRNA
??SNX16	??64089	People's sorting connects protein 16 (SNX16), transcribes variant 1, mRNA
			??SNX16	??64089	People's sorting connects protein 16 (SNX16), transcribes variant 2, mRNA
??SNX16	??64089	People's sorting connects protein 16 (SNX16), transcribes variant 3, mRNA
			??TNFRSF7	??939	The human tumor necrosis factor receptor superfamily, member 7 (TNFRSF7), mRNA
??ZAK	??51776	People's the kinases AZK (ZAK) that contains sterile α motif and leucine zipper, mRNA

Table 14

The product of the biomarker of mentioning in the table 13.What list is the Entrez gene I and the people RNA registration number and the human protein registration number of relevant reference sequences from Genbank.

Gene symbol	Gene I	The RNA registration number	The albumen registration number
				??CHD2	??1106	??NM_001271	??NP_001262
??CSPG6	??9126	??NM_005445	??NP_005436
				??CTSD	??1509	??NM_001909	??NP_001900
??FREB	??84824	??NM_032738	??NP_116127
				??GAS7	??8522	??NM_003644	??NP_003635
??GAS7	??8522	??NM_201432	??NP_958836
				??GAS7	??8522	??NM_201433	??NP_958839
??HIST1H1C	??3006	??NM_005319	??NP_005310
				??IGFBP7	??3490	??NM_001553	??NP_001544
??IRAK3	??11213	??NM_007199	??NP_009130
				??IREB2	??3658	??NM_004136	??NP_004127
??MLH3	??27030	??NM_014381	??NP_055196
				??MTHFS	??10588	??NM_006441	??NP_006432
??NELL2	??4753	??NM_006159	??NP_006150
				??PLDN	??26258	??NM_012388	??NP_036520
??RPS24	??6229	??NM_001026	??NP_001017
				??RPS24	??6229	??NM_033022	??NP_148982
??SNTB2	??6645	??NM_006750	??NP_006741
				??SNTB2	??6645	??NM_130845	??NP_570896
??SNX16	??64089	??NM_022133	??NP_071416
				??SNX16	??64089	??NM_152836	??NP_690049
??SNX16	??64089	??NM_152837	??NP_690050
				??TNFRSF7	??939	??NM_001242	??NP_001233
??ZAK	??51776	??NM_016653	??NP_057737
				??ZAK	??51776	??NM_133646	??NP_598407

Table 15

Be used for table 13 and mention the primer of the real-time quantitative RT-PCR of biomarker.The reference sequences and 5 ' and 3 ' primer and the amplicon size that have shown described primer amplification.

Table 16

20 bladder cancer patients and 14 there is not mention in the table 13 of bladder cancer individuality the real-time quantitative RT-PCR result summary relatively of biomarker.

Table 17

The commercially available antibody of the protein of the biomarker of mentioning in the anti-table 13 of the present invention.

Embodiment 5

Also can use the QuantiTect of Qiagen ^TMProbe RT-PCR system (production number 204343) is corresponding with gene of interest

Double-tagging probe and primer are implemented QRT PCR.Described Probe and primer can be from Applied BiosystemsAssays-On-Demand ^TMOrder.

The double-tagging probe contains fluorophor and quencher molecule.The approaching of described quencher molecule and fluorescence report group stoped reporter group to send fluorescence, but extends in the step at PCR, and 5 '-3 ' exonuclease activity of Taq archaeal dna polymerase has discharged fluorophor, makes it send fluorescence.So, the amount of fluorescence is relevant with the PCR product of generation.Be used for the product of the disclosed selected biomarker of table 13 according to the present invention

The example of probe is displayed in Table 18.

Embodiment 6

Real-time RT-PCR result's statistical study

To separating from being categorized as the statistical study that suffer from the bladder cancer individuality (or its stage) or the PCR in real time analysis of not suffering from the blood sample of bladder cancer individuality is to use currently known methods, to obtain the abundance level of corresponding biomarker of the present invention in T-group.

The colony of selecting to suffer from the bladder cancer individuality and not suffering from the bladder cancer individuality.Select similar individuality to distribute, for example phenotype such as age, sex, weight index (BMI).One-way analysis of variance is determined the selection of sample between use KW group, makes and relatively can make based on age and BMI that it can be understood by those skilled in the art.

Embodiment 7

Use the product of biomarker described in Fig. 1, the gene expression profile of contrast normal individual is analyzed and is suffered from wing The gene expression profile of the blood sample of Guang cancer individuality

This embodiment provides the invention of institute's prescription in the purposes of identifying on each biomarker of the present invention, described evaluation is to finish from differential gene expression in the blood sample of suffering from bladder cancer patients by comparing with the blood sample from healthy patients, detecting.

Blood sample is taken from the patient that clinical diagnosis is a bladder cancer defined herein; Patient and the one or more test patient of not suffering from bladder cancer defined herein.Then, analyze the gene expression profile of biomarker combination of the present invention, the gene expression profile of test genes of individuals express spectra and two kinds of contrasts compares.

Come total RNA of autothermic cracking whole blood to take from each patient, it is at first used

Reagent (GIBCO) separates, and generates the fluorescently-labeled probe at each blood sample then, and sex change also hybridizes to

On the U1332.0 version microarray, it contains the oligonucleotide corresponding to each gene described in the table 1.Detect the scanning and Scanalyzer software (the Michael Eisen that then pass through GMS scanner 418 with the specific hybrid of described array, Stanford University) processing of experimental data is measured, (SiliconGenetics CA) analyzes to use GeneSpring software then.Determine that by statistical study corresponding to the differential expression of RNA product between described two kinds of control populations and described test individuality of described biomarker, Wilcox Mann Whitney rank test (Glantz SA.Primerof Biostatistics.5 has been used in described statistical study in blood sample ^ThEd.New York, USA:McGraw-Hill Medical PublishingDivision, 2002).

Embodiment 8

The logistic recurrence is applied to combination of the present invention, is used to distinguish the rank of bladder cancer or bladder cancer with evaluation The sorter of Duan Yufei bladder cancer and combination

Use real-time quantitative RT-PCR (QRT-PCR) to determine corresponding to table 10, and/or the data of the RNA product level of biomarker in the table 13, it is at the QuantiTect that has used Qiagen in (T-group) ^TM

Green PCR test kit.In one case, described reference group is made up of the individuality of suffering from the bladder cancer stage of bladder cancer (no matter) and the individuality of not suffering from bladder cancer.In another case, described reference group forms (combination that is used to screen/diagnose early stage bladder cancer with evaluation) by the individuality of suffering from certain stage bladder cancer (for example early stage bladder cancer) and the individuality of not suffering from bladder cancer.Reference data set is made up of the Δ Ct value of each gene of each individuality among the reference group (being the Ct of RNA product of comparison sheet 10 biomarkers and the Ct of house-keeping gene), its data that can use the QRT-PCR of a group individuality to produce are set up, some suffer from bladder cancer in the described a group individuality, and some do not suffer from bladder cancer.Notice that described house-keeping gene as suggested in its name, is to suffer from bladder cancer and do not suffering from the gene that noticeable change on the statistics does not take place between the bladder cancer individuality.We notice, GAPDH still is that the β Actin muscle all can be used as house-keeping gene and uses, so another house-keeping gene should use, RPLP0 for example, B2M, RPS18 etc.The Logistic regression model can be applicable to each ten possible assortment of genes, and it uses the Δ Ct value that is produced.Therefore produce the sorter of form of the following equation 1 of gained.

X＝Logit(P)＝ln(P/(1-P))＝b ₀+b ₁ΔCt ₁+b ₂ΔCt ₂+...+b _nΔCt _n(Eq1)

Wherein P=patient's sample is diagnosed as the possibility of " ill " (for example suffering from bladder cancer).As referred, X=Logit (p) can be defined as follows:

If X 〉=threshold value, if Y=1 (diagnosis=" ill " for example suffer from bladder cancer) then is X＜threshold value, then Y=0 (diagnosis=" contrast ").As is understood, select described threshold value so that required specificity or susceptibility to be set.The susceptibility or the specificity of gained ROC area and maintenance are determined, and therefore providing described diagnosis is the similar measurement of true positives or false positive results.

Cross validation

Identify useful biomarker combination and these combinations had after the specific sorter that with aforesaid method can use the training group that the result is carried out cross validation, it uses one or more currently known methodss.For example, can use the WEKA (sorter of cross validation gained (22) of http://www.cs.waikato.ac.nz/～ml/weka/index.html).In one embodiment, can use two kinds of different cross validation configurations.For example, 100 bootstrap that WEKA MetaAnalysis function can be used for making up training data group and each new data set repeat, and it is to use simple logistic model analysis.Then, can analyze the result (" bootstrap aggregating ") of logistic equation (sorter) and all equations of equalization of each gained by 10 times of cross validations.

Prediction (blind) check

In order further to confirm and select the sorter of one or more gained, can check one group of blind clinical sample.Use is by contrast and suffer from the check group that the bladder cancer object is formed.Preferably, there is not blind test group individuality to be used to create reference data set.Can use the sorter of one or more gained to estimate the observed value and the cross validation of each blind sample, described sorter is defined by the training group.In addition, also can use the voting scheme of having used two or more gained sorters.For example, can use the algorithm of forming by initial calculation result, binary-logic gate and " council's machine " ballot. Initial calculation result: can calculate logit function X for each equation (I=1 is to N) _i, described equation provides ROC area＞for example with respect to reference data set, and 0.6,0.7,0.8 or 0.9. Logical gateIf: X _iThe threshold value of＜j (j represents the equation numbering herein) then must be divided into S for the blind sample of j equation _i=-1.If X _iThe threshold value of 〉=j then must be divided into S for the blind sample of j equation _i=+1. The ballot of council's machine (23): make ballot according to score then from 10 or more diagnostic logit equation.According to definition, ballot=∑ S _iIf ballot≤0 then sample is called as " no prostatitis gland cancer ", if ballot＞0 then sample is called as " ill or trouble bladder cancer ".

Embodiment 9

Two genes " ratio " of selected biomarker are used as mathematical model, are used to distinguish bladder thereby produce The sorter of cancer and non-bladder cancer

For fear of identifying house-keeping gene, taked the strategy of the gene differential expression of two expression of direct comparison, wonderful, confirm that it is more favourable than using house-keeping gene.Dual-gene ratio was described (16,17) before the application of distinguishing on the disease group, it has used the cDNA array data (rather than coming autoblood) that comes self-organization.Described technology has reduced between the individuality that biotic factor and technical factor cause and has changed, and also is independent of the expression measuring table that is used to obtain data.Other advantage of dual-gene method comprises the sample size that described method is relatively independent of input, and making does not need to use house-keeping gene to contrast as loading, and described method only needs a spot of RNA.At last, use the ratio of up-regulated gene and down-regulated gene, therefore the signal that increased makes mensuration more responsive.

As shown in table 10,5 biomarkers are accredited as rise between bladder cancer patients and control patients, and 5 biomarkers are accredited as downward modulation.In this embodiment, the combination of two biomarkers (rise, a downward modulation (is wherein raised and is referred to than the patient who does not suffer from bladder cancer, gene has the expression of higher level in bladder cancer patients, and downward modulation refers to than the patient who does not suffer from bladder cancer, gene has the expression of lower level in bladder cancer patients)) selected input logistic recurrence, to determine the diagnosis capability of difference to making up from the listed biomarker of table 10.In returning like logistic, maximal phase estimated a plurality of combinations, to determine separating capacity (ROC area＞0.5) to " bladder cancer " and " contrast ".

Table 18. real-time RT-PCR result has provided a plurality of two assortments of genes with classification capacity with differentiation bladder cancer and non-bladder cancer sample

The described centering that the training group who forms in use tests, the Δ Ct between IGFBP7 and the CHD2 is than putting up the best performance in distinguishing bladder cancer patients (n=20) and normal patient (n=14).Described right score is as follows: AUC is 0.907, susceptibility 80.0% (95%CI 56.3-94.1%), specificity 92.9% (95%CI 66.1-98.8%).Δ Ct ratio between IGFBP7 and the NELL2 comes second the best, and its AUC is 0.879, susceptibility 90.0% (95%CI 68.3-98.5%), specificity 85.7% (95%CI 57.2-97.8%).Then, this ratio of test in another training group who forms by more a plurality of bodies, promptly to cause the mensuration susceptibility in this group be 83%, correctly predicted 34 in 40 bladder cancer.In 6 bladder cancer patients 3 by the be cut patient of top layer bladder cancer of described mensuration error prediction for not suffering from bladder cancer.This prompting, described mensuration may be used for determining patient's prognosis.The specificity of described mensuration be have only in 93%, 27 normal healthy controls 2 mispredicted for suffering from bladder cancer.

Embodiment 9

Two genes " ratio " of selected biomarker are used as mathematical model, are used to distinguish bladder thereby produce The sorter of cancer and non-bladder cancer

More there not to be the useful combination of omitting ground identified gene ratio, these genes are used to use biomarker to suffer from the patient of bladder cancer with screening and/or diagnosis from the subclass of the mark of table 13 in selection.Selection is by 20 individuality and 14 reference training groups that the individuality of not suffering from bladder cancer is formed that suffer from bladder cancer.Reference data set is corresponding to selected biomarker SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, the Δ Ct ratio of the RNA product of NELL2 and CHD2.Therefore use the QuantiTect of real-time quantitative RT-PCR (QRT-PCR) and Qiagen ^TM

Green PCR test kit is determined CHD2/CTSD, CHD2/SNX16, CHD2/CSPG6, CHD2/IGFBP7, CHD2/TNFRSF7, CHD2/NELL2, NELL2/SNX16, NELL2/CSPG6, NELL2/IGFBP7, NELL2/CTSD, NELL2/TNFRSF7, TNFRSF7/SNX16, TNFRSF7/CSPG6, TNFRSF7/IGFBP7, TNFRSF7/CSTD, CTSD/SNX16, CTSD/CSPG6, CTSD/IGFBP7, IGFBP7/SNX16, the Δ Ct ratio of IGFBP7/CSPG6 and CSPG/SNX16 is wherein measured every couple Ct in single 96 orifice plates, so that compare the Ct value better.(note using multiplexed TaqMan probe can obtain similar result with band different colours fluorophor, it will be understood by those skilled in the art) come the identification of organism mark right in this way, allow value of input to represent the expression level of described gene pairs, the single item during it returns as logistic.Therefore, can create the combination of a ratio, two ratios, three ratios, four ratios, five ratios, six ratios or all ratios, make the general formula of gained logistic regression equation (sorter) be

X＝Logit(P)＝ln(P/(1-P))＝b ₀+b ₁ΔCt ₁+b ₂ΔCt ₂+...+b _nΔCt _n????(Eq1)

Wherein P=patient's sample is diagnosed as the possibility of " ill " (for example suffering from bladder cancer), and the form of single ratio combination is as follows:

X=Logit (P)=ln (P/ (1-P))=b ₀+ b ₁Δ Ct ₁(Eq2) Δ Ct wherein ₁Be data corresponding to any one above-mentioned ratio.

Perhaps the form of two ratio combinations is as follows:

X=Logit (P)=ln (P/ (1-P))=b ₀+ b ₁Δ Ct ₁+ Δ Ct ₂(Eq3) Δ Ct wherein ₁Be data corresponding to any one above-mentioned ratio, Δ Ct ₂Also be data corresponding to any one above-mentioned ratio.

Similarly, all three ratios combinations have been tested, combination of four ratios and the combination of five ratios.

Each ROC area＞0.5, the gained sorter greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, greater than 0.95 can be used for diagnosing or screening the test individuality of suffering from or not suffering from bladder cancer.Fig. 1-4 shows the graphic result of the sorter that is obtained by digital ratio value, two ratios, three ratios and four ratios.

Use 40 representative sorters of four ratios of suffering from the spread training group of bladder cancer individuality and 27 contrast individualities to be displayed in Table 19, table 20 shows the representative sorter of three ratios, table 21 shows the representative sorter of two ratios, and hereinafter table 22 shows the representative sorter of digital ratio value.

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Equivalent

Those skilled in the art only use normal experiment just will understand, maybe can determine, the equivalent of many particular of the present invention.These equivalents are also included within the following claim scope.

Sequence table

＜110〉applicant: Genenews Inc.

Liu Zongzheng

Han Xiaoyan

Rob Thomas-Jaeger

Zhao Chengen

Zheng Run

＜120〉denomination of invention: bladder cancer biomarkers and uses thereof

＜130〉document code: 204231/2148

＜140〉the application number: PCT/US06/16816

＜141〉the application's day: 2006-06-05

＜150〉application number formerly: 60/676,921

＜151〉applying date: 2005-05-02 formerly

＜150〉application number formerly: 60/729,056

＜151〉applying date: 2005-10-21 formerly

＜160〉sequence number: 780

＜170〉software: PatentIn version 3.3

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ttcccaggtg?gtctcattgg?t?????????????????????????????????????21

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tctggtgcaa?acgaaaggca???????????????????????????????????????20

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tggcttctta?ggtttggttc?cg????????????????????????????????????22

<210>SEQ?ID?NO?15

＜211〉length: 21

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＜223〉out of Memory: forward primer is used for registration number: NM_001553

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tgcgagcaag?gtccttccat?a????????????????????????21

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atgaggacag?gtgtcgggat?tc??????????????????????22

<210>SEQ?ID?NO?17

＜211〉length: 22

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＜213〉biology: artificial sequence

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＜223〉out of Memory: forward primer is used for registration number: NM_007199

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attgcagtgt?gtaggtgaca?cg??????????????????????22

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＜223〉out of Memory: antisense sequences is used for registration number: NM_007199

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agcatggttg?aacgttgtgc?ag?????????????????????22

<210>SEQ?ID?NO?19

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＜223〉out of Memory: forward primer is used for registration number: NM_004136

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accagtgcct?gaacctgaaa?ca?????????????????????22

<210>SEQ?ID?NO?20

＜211〉length: 21

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＜223〉out of Memory: reverse primer is used for registration number: NM_004136

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aggagggatc?actgccacat?t?????????????????????21

<210>SEQ?ID?NO?21

＜211〉length: 22

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＜223〉out of Memory: forward primer is used for registration number: NM_005784 and NM_014381

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acacggagga?tgacttgact?ga????????????????????22

<210>SEQ?ID?NO?22

＜211〉length: 21

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＜400〉sequence: 22

aaactggctt?ggctggacct?t?????????????????????21

<210>SEQ?ID?NO?23

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: forward primer is used for registration number: NM_006441

＜400〉sequence: 23

agcctggtga?gggtgatgtt????????????????????????????20

<210>SEQ?ID?NO?24

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: reverse primer is used for registration number: NM_006441

＜400〉sequence: 24

ccagtcggtt?gccatgtttg?t??????????????????????????21

<210>SEQ?ID?NO?25

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: forward primer is used for registration number: NM_006159

＜400〉sequence: 25

ccagctgtga?aacggacatt?ga????????????????????????22

<210>SEQ?ID?NO?26

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: reverse primer is used for registration number: NM_006159

＜400〉sequence: 26

tcatggtagc?catctctgca?ct????????????????????????22

<210>SEQ?ID?NO?27

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: forward primer is used for registration number: NM_012388

＜400〉sequence: 27

agccgacgcc?tggtttaagt?ga????????????????????????22

<210>SEQ?ID?NO?28

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: reverse primer is used for registration number: NM_012388

＜400〉sequence: 28

agcaatcctt?ctgccagttg?ct????????????????????????22

<210>SEQ?ID?NO?29

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: forward primer is used for registration number: NM_001026 and NM_033022

＜400〉sequence: 29

gcaacgaaag?gaacgcaaga?ac????????????????????????22

<210>SEQ?ID?NO?30

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: reverse primer is used for registration number: NM_001026 and NM_033022

＜400〉sequence: 30

actccttcgg?ctgtgatcca?at????????????????????????22

<210>SEQ?ID?NO?31

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 31

aagacaaaga?aggggacaag?g?????????????????????????21

<210>SEQ?ID?NO?32

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 32

caaaagccaa?gaggaggaca????????????????????????????20

<210>SEQ?ID?NO?33

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 33

gatgatgaca?agaagccaaa?gc?????????????????????????22

<210>SEQ?ID?NO?34

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 34

aggaaagaca?aagaagggga?ca?????????????????????????22

<210>SEQ?ID?NO?35

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 35

caaagcgatc?tcagggtcct????????????????????????????20

<210>SEQ?ID?NO?36

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 36

aatcttctga?gagtcagtcg?ga?????????????????????????22

<210>SEQ?ID?NO?37

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 37

agaagatgaa?caggaacaag?gc?????????????????????????22

<210>SEQ?ID?NO?38

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 38

tcagaagcct?catttgcct?????????????????????????????19

<210>SEQ?ID?NO?39

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 39

ggggaatcga?gtgcttatct?tc?????????????????????????22

<210>SEQ?ID?NO?40

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 40

ctggggattt?atgaacatgg?c????????????????????????????21

<210>SEQ?ID?NO?41

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 41

acaggttcac?ttcctgctgt?aa??????????????????????????22

<210>SEQ?ID?NO?42

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 42

tggatcactt?ccctgctca??????????????????????????????19

<210>SEQ?ID?NO?43

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 43

cgttcaagag?ggagaccaa??????????????????????????????19

<210>SEQ?ID?NO?44

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 44

gacaggttca?cttcctgctg?taa?????????????????????????23

<210>SEQ?ID?NO?45

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 45

agccccttgt?caggtttgt?????????????????????????????19

<210>SEQ?ID?NO?46

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 46

cttgtctgct?tcggtttgac????????????????????????????20

<210>SEQ?ID?NO?47

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 47

taacacgagg?tttgggca?????????????????????????????18

<210>SEQ?ID?NO?48

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 48

ttgttgccac?caccatagtt?g????????????????????21

<210>SEQ?ID?NO?49

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 49

tttctccctt?gatggaacca??????????????????????20

<210>SEQ?ID?NO?50

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001271

＜400〉sequence: 50

cttcaacaag?taatccgctc?g????????????????????21

<210>SEQ?ID?NO?51

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 51

ggtgatgcca?aatcttcgag?ta??????????????????22

<210>SEQ?ID?NO?52

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 52

ttcgggttca?gactcaggca?????????????????????20

<210>SEQ?ID?NO?53

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 53

aaggacctcg?tggagggatt?t??????????????????21

<210>SEQ?ID?NO?54

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 54

gtggtgatgc?caaatcttcg?ag?????????????????22

<210>SEQ?ID?NO?55

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 55

cgtgaaaaag?gcactgaaac?agct???????????????24

<210>SEQ?ID?NO?56

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe

＜400〉sequence: 56

gttccaaatc?ccagccagtc?ct?????????????????22

<210>SEQ?ID?NO?57

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 57

agtaaaagcc?agaagacctg?tcc????????????????????????????23

<210>SEQ?ID?NO?58

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 58

ccccaacaag?agacacttca?gt?????????????????????????????22

<210>SEQ?ID?NO?59

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 59

ctcagatggt?gagaatgttg?ga?????????????????????????????22

<210>SEQ?ID?NO?60

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001271

＜400〉sequence: 60

taaaaagcct?caggggaagc????????????????????????????????20

<210>SEQ?ID?NO?61

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 61

tcacaaagca?gtgtcccatc????????????????????????????????20

<210>SEQ?ID?NO?62

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 62

cacatgcgtg?gaagtcactg????????????????????????????????20

<210>SEQ?ID?NO?63

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 63

aggggttctg?gctcacaaa????????????????????????????????19

<210>SEQ?ID?NO?64

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 64

tgctagacca?cttccgtcga???????????????????????????????20

<210>SEQ?ID?NO?65

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 65

ccgtcgaaaa?ggaataaacc?ag????????????????????22

<210>SEQ?ID?NO?66

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 66

agcatggaag?tttcaaccca???????????????????????20

<210>SEQ?ID?NO?67

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 67

gagagagaca?gaagggggta?ctgt?????????????????24

<210>SEQ?ID?NO?68

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 68

tgagattcgt?caacttcagc?ag???????????????????22

<210>SEQ?ID?NO?69

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 69

acaaagcagt?gtcccatcag?????????????????????20

<210>SEQ?ID?NO?70

＜211〉length: 26

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 70

gttagatgtc?agggatacag?cctatc??????????????26

<210>SEQ?ID?NO?71

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 71

agcaagggct?accaaggatt????????????????????20

<210>SEQ?ID?NO?72

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 72

gataggctgt?atccctgaca?tcta???????????????24

<210>SEQ?ID?NO?73

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 73

tcagagcaag?ggctaccaa????????????????????????????????19

<210>SEQ?ID?NO?74

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 74

tcttcgtgct?gacttcatct?ga????????????????????????????22

<210>SEQ?ID?NO?75

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 75

tcttcgtgct?gacttcatct?g?????????????????????????????21

<210>SEQ?ID?NO?76

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 76

tccttgtgtc?ataataaccc?cc????????????????????????????22

<210>SEQ?ID?NO?77

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 77

ctgaagctcc?ttaattccag?ct????????????????????????????22

<210>SEQ?ID?NO?78

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 78

tacccccttc?tgtctctctc?ag????????????????????????????22

<210>SEQ?ID?NO?79

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 79

gggctaccaa?ggatttctgt?c????????????????????????????21

<210>SEQ?ID?NO?80

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005445

＜400〉sequence: 80

atagtgaaag?cacgggcca???????????????????????????????19

<210>SEQ?ID?NO?81

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 81

gagaaatgca?acagctttca?gg???????????????????????????22

<210>SEQ?ID?NO?82

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 82

cctggagagg?ttacttttct?gcc????????????????????????????23

<210>SEQ?ID?NO?83

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 83

aatgcaacag?ctttcaggtg?g??????????????????????????????21

<210>SEQ?ID?NO?84

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 84

acatgcgtgg?aagtcactgc?t??????????????????????????????21

<210>SEQ?ID?NO?85

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 85

atgcgtggaa?gtcactgctg????????????????????????????????20

<210>SEQ?ID?NO?86

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 86

gcccgtgctt?tcactatgga???????????????????????????????20

<210>SEQ?ID?NO?87

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 87

agacactatg?gcacgatcag?aag???????????????????????????23

<210>SEQ?ID?NO?88

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 88

atctgagaaa?acgcttggac?c?????????????????????????????21

<210>SEQ?ID?NO?89

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 89

gagaaatgca?acagctttca?gg????????????????????????????22

<210>SEQ?ID?NO?90

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005445

＜400〉sequence: 90

tgtcgtagca?tggaagtttc?aacc????????????????????????????????24

<210>SEQ?ID?NO?91

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 91

ggtggcacag?actccaagta?tt??????????????????????????????????22

<210>SEQ?ID?NO?92

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 92

tgcttcacag?tcgtcttcg??????????????????????????????????????19

<210>SEQ?ID?NO?93

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 93

ggaggacctg?attgccaaag????????????????????????????????????20

<210>SEQ?ID?NO?94

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 94

ggtggcacag?actccaagta?t??????????????????????????????????21

<210>SEQ?ID?NO?95

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 95

gcttcacagt?cgtcttcgac?a??????????????????????????????????21

<210>SEQ?ID?NO?96

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 96

cgaggtgctc?aagaactaca?tg?????????????????????????????????22

<210>SEQ?ID?NO?97

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 97

aaaggccccg?tctcaaagta????????????????????????????????????20

<210>SEQ?ID?NO?98

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 98

gtgcttcaca?gtcgtcttcg????????????????????????????????????20

<210>SEQ?ID?NO?99

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 99

attcccgagg?tgctcaaga????????????????????????????????????19

<210>SEQ?ID?NO?100

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 100

aaggccccgt?ctcaaagta????????????????????????????????????19

<210>SEQ?ID?NO?101

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 101

atgagggaag?tgcctgtgtc???????????????????????????????????20

<210>SEQ?ID?NO?102

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 102

gctggacttg?tcgctgttgt???????????????????????????????????20

<210>SEQ?ID?NO?103

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 103

tgtcgaagac?gactgtgaag?c????????????????????????????????21

<210>SEQ?ID?NO?104

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 104

catgagggaa?gtgcctgtgt??????????????????????????????????20

<210>SEQ?ID?NO?105

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 105

taggtgctgg?acttgtcgct??????????????????????????????????20

<210>SEQ?ID?NO?106

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 106

gatgtccagc?agtttgcagt??????????????????????????????????20

<210>SEQ?ID?NO?107

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 107

cgtgtcgaag?acgactgtga????????????????????????????????20

<210>SEQ?ID?NO?108

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 108

gccatagtgg?atgtcaaacg?a??????????????????????????????21

<210>SEQ?ID?NO?109

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 109

tccagcagtt?tgcagtgga????????????????????????????????19

<210>SEQ?ID?NO?110

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001909

＜400〉sequence: 110

cgaagacgac?tgtgaagcac?t?????????????????????????????21

<210>SEQ?ID?NO?111

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 111

caagggttct?ctgtcctacc?tga???????????????????????????23

<210>SEQ?ID?NO?112

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 112

ccatccactg?caaactgctg?ga????????????????????????????22

<210>SEQ?ID?NO?113

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 113

tactacgggg?agattggcat?cg????????????????????????????22

<210>SEQ?ID?NO?114

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 114

acaagggttc?tctgtcctac?ctga??????????????????????????24

<210>SEQ?ID?NO?115

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 115

tccactgcaa?actgctggac?atc????????????????????????????????23

<210>SEQ?ID?NO?116

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 116

tcacagtcgt?cttcgacacg????????????????????????????????????20

<210>SEQ?ID?NO?117

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 117

tactacgggg?agattggcat?c??????????????????????????????????21

<210>SEQ?ID?NO?118

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 118

tcccctccat?ccactgcaaa????????????????????????????????????20

<210>SEQ?ID?NO?119

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 119

tcacagtcgt?cttcgacacg????????????????????????????????????20

<210>SEQ?ID?NO?120

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001909

＜400〉sequence: 120

ttcccgaggt?gctcaagaac?t??????????????????????????????????21

<210>SEQ?ID?NO?121

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 121

acttgactga?tgcaagggaa????????????????????????????????????20

<210>SEQ?ID?NO?122

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 122

cacagtcacc?atgaagctgg????????????????????????????????????20

<210>SEQ?ID?NO?123

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 123

cctctacctt?tcccttggtg?t????????????????????????????????????21

<210>SEQ?ID?NO?124

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 124

tcactccggg?tcatactggt??????????????????????????????????????20

<210>SEQ?ID?NO?125

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 125

cacacggagg?atgacttgac?t????????????????????????????????????21

<210>SEQ?ID?NO?126

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 126

ctctaccttt?cccttggtgt?g????????????????????????????????????21

<210>SEQ?ID?NO?127

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 127

acttgactga?tgcaagggaa?g????????????????????????????????????21

<210>SEQ?ID?NO?128

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 128

ttgtggctat?cacagtccaa?g????????????????????????????????????21

<210>SEQ?ID?NO?129

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 129

ggggttgcag?gagacctaaa??????????????????????????????????????20

<210>SEQ?ID?NO?130

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 130

gaggatgact?tgactgatgc?a????????????????????????????????????21

<210>SEQ?ID?NO?131

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 131

aaaactggct?tggctggac??????????????????????????????????????19

<210>SEQ?ID?NO?132

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 132

catcagtcaa?gtcatcctcc?g????????????????????????????????????????21

<210>SEQ?ID?NO?133

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 133

gcatcagtca?agtcatcctc?c????????????????????????????????????????21

<210>SEQ?ID?NO?134

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 134

tctgaggagc?tggattcaat?gt???????????????????????????????????????22

<210>SEQ?ID?NO?135

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 135

gtccccttca?aaaactggct?t????????????????????????????????????????21

<210>SEQ?ID?NO?136

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 136

cttcccttgc?atcagtcaag?t????????????????????????????????????????21

<210>SEQ?ID?NO?137

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 137

agcaggtccc?cttcaaaaac?t????????????????????????????????????????21

<210>SEQ?ID?NO?138

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 138

ttgtagaagg?agaagaggag?gc???????????????????????????????????????22

<210>SEQ?ID?NO?139

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 139

tgcagcgtct?caaaactgg??????????????????????????????????????????19

<210>SEQ?ID?NO?140

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_032738

＜400〉sequence: 140

gtccccttca?aaaactggc????????????????????????????????????????19

<210>SEQ?ID?NO?141

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 141

tcagtgaacc?cttccacctg?at????????????????????????????????????22

<210>SEQ?ID?NO?142

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 142

gccagttttg?agacgctgca??????????????????????????????????????20

<210>SEQ?ID?NO?143

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 143

gccagttttg?agacgctgca?????????????????????????????????????20

<210>SEQ?ID?NO?144

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 144

ttggaaacag?agcccccagc?ta?????????????????????????????????22

<210>SEQ?ID?NO?145

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 145

tcagtgaacc?cttccacctg????????????????????????????????????20

<210>SEQ?ID?NO?146

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 146

gccagttttg?agacgctgca???????????????????????????????????20

<210>SEQ?ID?NO?147

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 147

tcagtgaacc?cttccacctg???????????????????????????????????20

<210>SEQ?ID?NO?148

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 148

actgtttcca?gcgccaattc?tc????????????????????????????22

<210>SEQ?ID?NO?149

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 149

tcaccatgaa?gctgggctgt?gt????????????????????????????22

<210>SEQ?ID?NO?150

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_032738

＜400〉sequence: 150

cagtgaaccc?ttccacctga??????????????????????????????20

<210>SEQ?ID?NO?151

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 151

gctgagcaac?aagacagagg?ag???????????????????????????22

<210>SEQ?ID?NO?152

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 152

tcgccaagca?aaaagcag????????????????????????????????18

<210>SEQ?ID?NO?153

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 153

tgataagaag?gacccccaag??????????????????????????????20

<210>SEQ?ID?NO?154

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 154

ggagaacagc?tttgacgatg?t????????????????????????????21

<210>SEQ?ID?NO?155

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 155

agaactcctt?ggcttcacag?g????????????????????????????21

<210>SEQ?ID?NO?156

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 156

tcatgcgctg?tgtggatct???????????????????????????????19

<210>SEQ?ID?NO?157

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 157

gagcaaggaa?aacaccatca?c????????????????????????????????21

<210>SEQ?ID?NO?158

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 158

aagaagtgcg?accaccacat?t????????????????????????????????21

<210>SEQ?ID?NO?159

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 159

gctgagcaac?aagacagagg?ag???????????????????????????????22

<210>SEQ?ID?NO?160

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 160

ctgaaaccaa?ccgagtgga??????????????????????????????????19

<210>SEQ?ID?NO?161

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 161

atttggactg?ggcctggtt??????????????????????????????????19

<210>SEQ?ID?NO?162

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 162

ccttgggggt?ccttcttatc????????????????????????????????20

<210>SEQ?ID?NO?163

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 163

aagccaagga?gttctgagag?ag?????????????????????????????22

<210>SEQ?ID?NO?164

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 164

cactcggttg?gtttcagcag???????????????????????????????20

<210>SEQ?ID?NO?165

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 165

ggaagttcat?caggggctt?????????????????????????????????????19

<210>SEQ?ID?NO?166

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 166

atcatctcta?ccctctccac?ct?????????????????????????????????22

<210>SEQ?ID?NO?167

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 167

cagtagttca?aacccagcca????????????????????????????????????20

<210>SEQ?ID?NO?168

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 168

tgtcctcctc?tgtcttgttg?c??????????????????????????????????21

<210>SEQ?ID?NO?169

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 169

ctcttcaaac?catttggact?gg?????????????????????????????????22

<210>SEQ?ID?NO?170

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_003644

＜400〉sequence: 170

tccttctgca?tttgtttgcc????????????????????????????????????20

<210>SEQ?ID?NO?171

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 171

acctcatgcg?ctgtgtggat????????????????????????????????????20

<210>SEQ?ID?NO?172

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 172

agctgctgaa?accaaccgag?t??????????????????????????????????21

<210>SEQ?ID?NO?173

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 173

tggctgggtt?tgaactactg?c????????????????????????????????21

<210>SEQ?ID?NO?174

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 174

gagcaaggaa?aacaccatca?ca???????????????????????????????22

<210>SEQ?ID?NO?175

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 175

tcacctcaag?ttctctgcca?a????????????????????????????????21

<210>SEQ?ID?NO?176

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 176

gcccagtcca?aatggtttga??????????????????????????????????20

<210>SEQ?ID?NO?177

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 177

agctgctgaa?accaaccgag?t????????????????????????????????21

<210>SEQ?ID?NO?178

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 178

ctggagatga?agacccagca??????????????????????????????????20

<210>SEQ?ID?NO?179

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 179

tcatgcgctg?tgtggatctc???????????????????????????????????20

<210>SEQ?ID?NO?180

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_003644

＜400〉sequence: 180

accgtggctg?ggtttgaact?ac????????????????????????????????22

<210>SEQ?ID?NO?181

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 181

tcacctcaag?ttctctgcca???????????????????????????????????20

<210>SEQ?ID?NO?182

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 182

tcaagctgag?caacaagaca?g????????????????????????????????????????21

<210>SEQ?ID?NO?183

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 183

tgagtgtccg?aaaatccacc??????????????????????????????????????????20

<210>SEQ?ID?NO?184

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 184

tgcgctgtgt?ggatctcta??????????????????????????????????????????19

<210>SEQ?ID?NO?185

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 185

ctgaaaccaa?ccgagtggag?????????????????????????????????????????20

<210>SEQ?ID?NO?186

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 186

cagagcaagg?aaaacaccat?c???????????????????????????????????????21

<210>SEQ?ID?NO?187

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 187

aagtgcgacc?accacattg?????????????????????????????????????????19

<210>SEQ?ID?NO?188

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 188

cagagcaagg?aaaacaccat?c??????????????????????????????????????21

<210>SEQ?ID?NO?189

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 189

gcagagcaag?gaaaacacca????????????????????????????????????????20

<210>SEQ?ID?NO?190

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 190

aacaagacag?aggaggacat?ca????????????????????????22

<210>SEQ?ID?NO?191

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 191

atgtggtggt?cgcacttct????????????????????????????19

<210>SEQ?ID?NO?192

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 192

tcaaaccatt?tggactggg????????????????????????????19

<210>SEQ?ID?NO?193

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 193

tccactcggt?tggtttcag????????????????????????????19

<210>SEQ?ID?NO?194

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 194

ggatcatctc?taccctctcc?a?????????????????????????21

<210>SEQ?ID?NO?195

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 195

catttgtttg?cccttcagct???????????????????????????20

<210>SEQ?ID?NO?196

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 196

ctggagcagt?agttcaaacc?c?????????????????????????21

<210>SEQ?ID?NO?197

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 197

gtcctcctct?gtcttgttgc?t?????????????????????????21

<210>SEQ?ID?NO?198

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 198

ccttgggggt?ccttcttatc????????????????????????????????20

<210>SEQ?ID?NO?199

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 199

ggagcagtag?ttcaaaccca?g?????????????????????????????21

<210>SEQ?ID?NO?200

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201432

＜400〉sequence: 200

tgctgccgga?tcatctcta????????????????????????????????19

<210>SEQ?ID?NO?201

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 201

ttcacagcga?ggtggagaag???????????????????????????????20

<210>SEQ?ID?NO?202

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 202

acctcatgcg?ctgtgtggat??????????????????????????????20

<210>SEQ?ID?NO?203

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 203

tcgccaagca?aaaagcagag??????????????????????????????20

<210>SEQ?ID?NO?204

＜211〉length: 25

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 204

aggcccagtc?caaatggttt?gaaga????????????????????????25

<210>SEQ?ID?NO?205

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 205

gggtttgaac?tactgctcca?ga???????????????????????????22

<210>SEQ?ID?NO?206

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 206

agctgctgaa?accaaccgag?t????????????????????????????21

<210>SEQ?ID?NO?207

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 207

gacctggaga?tgaagaccca????????????????????????????????20

<210>SEQ?ID?NO?208

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 208

agctgctgaa?accaaccgag?t?????????????????????????????21

<210>SEQ?ID?NO?209

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 209

agctgctgaa?accaaccgag?t?????????????????????????????21

<210>SEQ?ID?NO?210

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201432

＜400〉sequence: 210

tctacaacca?ggcccagtcc?aaa??????????????????????????23

<210>SEQ?ID?NO?211

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 211

gcaaggaaaa?caccatcaca?????????????????????????????20

<210>SEQ?ID?NO?212

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 212

ctgaaaccaa?ccgagtgga??????????????????????????????19

<210>SEQ?ID?NO?213

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 213

tcaagctgag?caacaagaca?g????????????????????????????21

<210>SEQ?ID?NO?214

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 214

gagcaaggaa?aacaccatca?????????????????????????????20

<210>SEQ?ID?NO?215

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 215

aacaagacag?aggaggacat?ca????????????????????????22

<210>SEQ?ID?NO?216

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 216

cacctcaagt?tctctgccaa??????????????????????????20

<210>SEQ?ID?NO?217

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 217

acaagacaga?ggaggacatc?aag??????????????????????23

<210>SEQ?ID?NO?218

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 218

tgagtgtccg?aaaatccacc??????????????????????????20

<210>SEQ?ID?NO?219

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 219

acctggagat?gaagacccag??????????????????????????20

<210>SEQ?ID?NO?220

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 220

atgaagaagt?gcgaccacca??????????????????????????20

<210>SEQ?ID?NO?221

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 221

ggagcagtag?ttcaaaccca?g????????????????????????21

<210>SEQ?ID?NO?222

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 222

catttgtttg?cccttcagct??????????????????????????20

<210>SEQ?ID?NO?223

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 223

ctcttcaaac?catttggact?gg????????????????????????22

<210>SEQ?ID?NO?224

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 224

gtttctggag?cagtagttca?aacc?????????????????????24

<210>SEQ?ID?NO?225

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 225

aaccatttgg?actgggcct??????????????????????????19

<210>SEQ?ID?NO?226

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 226

atgtggtggt?cgcacttctt????????????????????????20

<210>SEQ?ID?NO?227

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 227

tgctgccgga?tcatctctac????????????????????????20

<210>SEQ?ID?NO?228

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 228

agaagtagtc?gcagtagctc?cact???????????????????24

<210>SEQ?ID?NO?229

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 229

caaaccattt?ggactgggc????????????????????????19

<210>SEQ?ID?NO?230

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_201433

＜400〉sequence: 230

atgtcctcct?ctgtcttgtt?gct???????????????????23

<210>SEQ?ID?NO?231

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 231

gctgctgaaa?ccaaccgagt???????????????????????20

<210>SEQ?ID?NO?232

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 232

tggctgggtt?tgaactactg?ctcc????????????????????????????24

<210>SEQ?ID?NO?233

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 233

tcatgcgctg?tgtggatctc????????????????????????????????20

<210>SEQ?ID?NO?234

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 234

gctgctgaaa?ccaaccgagt????????????????????????????????20

<210>SEQ?ID?NO?235

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 235

acctcatgcg?ctgtgtggat????????????????????????????????20

<210>SEQ?ID?NO?236

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 236

ttcacagcga?ggtggagaag????????????????????????????????20

<210>SEQ?ID?NO?237

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 237

acctcatgcg?ctgtgtggat?ct?????????????????????????????22

<210>SEQ?ID?NO?238

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 238

cagagcaagg?aaaacaccat?ca?????????????????????????????22

<210>SEQ?ID?NO?239

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 239

acctcatgcg?ctgtgtggat????????????????????????????????20

<210>SEQ?ID?NO?240

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_201433

＜400〉sequence: 240

attgccgacc?ttcgcaagca????????????????????20

<210>SEQ?ID?NO?241

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 241

gagctgtgag?gtcatcggaa?t??????????????????21

<210>SEQ?ID?NO?242

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 242

caggtgtact?tgagctgtga?ggt???????????????23

<210>SEQ?ID?NO?243

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 243

acagaactcc?tgcctggtga???????????????????20

<210>SEQ?ID?NO?244

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 244

tacttgagct?gtgaggtcat?cg????????????????22

<210>SEQ?ID?NO?245

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 245

gcatgaagta?actggctggg?t?????????????????21

<210>SEQ?ID?NO?246

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 246

agcatgaagt?aactggctgg?g????????????????21

<210>SEQ?ID?NO?247

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 247

gaagtaactg?gctgggtgct?????????????????20

<210>SEQ?ID?NO?248

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 248

gcatgaagta?actggctggg????????????????????????????????20

<210>SEQ?ID?NO?249

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 249

aaagcatgaa?gtaactggct?gg?????????????????????????????22

<210>SEQ?ID?NO?250

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 250

ggcccagaaa?agcatgaagt????????????????????????????????20

<210>SEQ?ID?NO?251

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 251

gcacccagcc?agttacttca????????????????????????????????20

<210>SEQ?ID?NO?252

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 252

gcacccagcc?agttacttca???????????????????????????????20

<210>SEQ?ID?NO?253

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 253

ccttgggaat?tggatgca????????????????????????????????18

<210>SEQ?ID?NO?254

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 254

ccagccagtt?acttcatgct?t????????????????????????????21

<210>SEQ?ID?NO?255

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 255

catgtaaggc?atcaaccact?g????????????????????????????21

<210>SEQ?ID?NO?256

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 256

tgatgctgaa?gcctgtcct??????????????????????????????19

<210>SEQ?ID?NO?257

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 257

catgtaaggc?atcaaccact?g????????????????????????????????21

<210>SEQ?ID?NO?258

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 258

ctgatgctga?agcctgtcct?????????????????????????????????20

<210>SEQ?ID?NO?259

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 259

tgctgatgct?gaagcctgt?????????????????????????????????19

<210>SEQ?ID?NO?260

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001553

＜400〉sequence: 260

ctgatgctga?agcctgtcct????????????????????????????????20

<210>SEQ?ID?NO?261

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 261

ggggtcacta?tggagttcaa?agg????????????????????????????23

<210>SEQ?ID?NO?262

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 262

ggggtcacta?tggagttcaa?agg????????????????????????????23

<210>SEQ?ID?NO?263

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 263

aagtaactgg?ctgggtgctg????????????????????????????????20

<210>SEQ?ID?NO?264

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 264

ggggtcacta?tggagttcaa?agg????????????????????????????23

<210>SEQ?ID?NO?265

＜211〉length: 23

IP0710943P. sequence table (official)

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 265

tgcatccaat?tcccaaggac?agg????????????????????????????23

<210>SEQ?ID?NO?266

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 266

cctctaagta?aggaagatgc?tgga??????????????????????????24

<210>SEQ?ID?NO?267

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 267

gcatccaatt?cccaaggaca?gg????????????????????????????22

<210>SEQ?ID?NO?268

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 268

cctctaagta?aggaagatgc?tgga??????????????????????????24

<210>SEQ?ID?NO?269

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 269

cctctaagta?aggaagatgc?tgga??????????????????????????24

<210>SEQ?ID?NO?270

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001553

＜400〉sequence: 270

cctctaagta?aggaagatgc?tgga??????????????????????????24

<210>SEQ?ID?NO?271

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 271

ctgccaagct?cttctgtttg???????????????????????????????20

<210>SEQ?ID?NO?272

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 272

tcggtcatct?gtggcagtat?a?????????????????????????????21

<210>SEQ?ID?NO?273

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 273

ttcaaccatg?ctcggtca????????????????????????????18

<210>SEQ?ID?NO?274

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 274

agccattcac?tacctgcaca?a????????????????????????21

<210>SEQ?ID?NO?275

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 275

ttcaaccatg?ctcggtca????????????????????????????18

<210>SEQ?ID?NO?276

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 276

gcgggcaaag?ttaagacca???????????????????????????19

<210>SEQ?ID?NO?277

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 277

accatcggtg?accttttaca?g????????????????????????21

<210>SEQ?ID?NO?278

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 278

gccaagctct?tctgtttgg??????????????????????????19

<210>SEQ?ID?NO?279

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 279

gccattcact?acctgcacaa????????????????????????20

<210>SEQ?ID?NO?280

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 280

aaccatgctc?ggtcatctg?????????????????????????19

<210>SEQ?ID?NO?281

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 281

gagaaggaca?cctgaaggac?ttt????????????????????23

<210>SEQ?ID?NO?282

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 282

tgctgctgct?ggtcatattt????????????????????????????20

<210>SEQ?ID?NO?283

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 283

tttactgctg?ctgctggtca??????????????????????????20

<210>SEQ?ID?NO?284

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 284

acaactctga?tgttctaggt?ggga????????????????????24

<210>SEQ?ID?NO?285

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 285

tgtttactgc?tgctgctgg?????????????????????????19

<210>SEQ?ID?NO?286

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 286

ccaggaatag?aggagaagga?ca????????????????????22

<210>SEQ?ID?NO?287

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 287

ttatccacgg?tgacattggc??????????????????????20

<210>SEQ?ID?NO?288

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 288

tggtacattc?tccaggaata?gagg????????????????24

<210>SEQ?ID?NO?289

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 289

caactctgat?gttctaggtg?gga?????????????????23

<210>SEQ?ID?NO?290

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_007199

＜400〉sequence: 290

atgtttactg?ctgctgctgg?t????????????????????????21

<210>SEQ?ID?NO?291

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 291

cgggcaaagt?taagaccatc?aa???????????????????????22

<210>SEQ?ID?NO?292

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 292

acttccggtc?ccacctagaa??????????????????????????20

<210>SEQ?ID?NO?293

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 293

acttccggtc?ccacctagaa??????????????????????????20

<210>SEQ?ID?NO?294

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 294

gctcggtcat?ctgtggcagt?at???????????????????????22

<210>SEQ?ID?NO?295

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 295

acttccggtc?ccacctagaa??????????????????????????20

<210>SEQ?ID?NO?296

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 296

gccagcttgt?attttgctga?ag???????????????????????22

<210>SEQ?ID?NO?297

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 297

gatgggacat?cgtcgagcta??????????????????????????20

<210>SEQ?ID?NO?298

＜211〉length: 26

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 298

gccagcttgt?attttgctga?agatcc????????????????????????????????26

<210>SEQ?ID?NO?299

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 299

ctcggtcatc?tgtggcagta?t????????????????????????????????????21

<210>SEQ?ID?NO?300

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_007199

＜400〉sequence: 300

acttccggtc?ccacctagaa?????????????????????????????????????20

<210>SEQ?ID?NO?301

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 301

acctgcatga?tatttggcct????????????????????????????????????20

<210>SEQ?ID?NO?302

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 302

tggaattggc?atagctccac????????????????????????????????????20

<210>SEQ?ID?NO?303

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 303

tgcaatccat?ctgtcatgc????????????????????????????????????19

<210>SEQ?ID?NO?304

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 304

gcaaagccaa?actcgaatc????????????????????????????????????19

<210>SEQ?ID?NO?305

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 305

tggggaataa?acggtggaa????????????????????????????????????19

<210>SEQ?ID?NO?306

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 306

aggaaactcc?agagactggg???????????????????????????????????20

<210>SEQ?ID?NO?307

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 307

tgttgaagct?ggtctgcgt????????????????????????????????????19

<210>SEQ?ID?NO?308

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 308

cacttcagtt?ccttccagga?ga????????????????????????????????22

<210>SEQ?ID?NO?309

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 309

gttatgcttg?gtctgccagt?tt????????????????????????????????22

<210>SEQ?ID?NO?310

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 310

ctgcccgtgt?tcttcttca????????????????????????????????????19

<210>SEQ?ID?NO?311

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 311

tgaatccggt?gcttctaagg??????????????????????????????????20

<210>SEQ?ID?NO?312

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 312

aacgaagcaa?tcacgctgaa?????????????????????????????????20

<210>SEQ?ID?NO?313

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 313

cccactgcct?ggagataaac?t??????????????????????????????21

<210>SEQ?ID?NO?314

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 314

tctatcctga?ggtctttttg?gacc??????????????????????????24

<210>SEQ?ID?NO?315

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 315

ttcaatagcc?tggagtgcaa????????????????????????????????????20

<210>SEQ?ID?NO?316

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 316

actgaagtgg?agctatgcca?at?????????????????????????????????22

<210>SEQ?ID?NO?317

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 317

catccatagc?caacgatttc???????????????????????????????????20

<210>SEQ?ID?NO?318

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 318

cgaagcaatc?acgctgaat????????????????????????????????????19

<210>SEQ?ID?NO?319

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 319

actttccagc?cactcctact?tg????????????????????????????????22

<210>SEQ?ID?NO?320

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_004136

＜400〉sequence: 320

tttctcagga?tcacctccaa?ga????????????????????????????????22

<210>SEQ?ID?NO?321

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 321

tggggaataa?acggtggaat??????????????????????????????????20

<210>SEQ?ID?NO?322

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 322

cctgaagaac?tgtctcctgg?aa???????????????????????????????22

<210>SEQ?ID?NO?323

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 323

gttgaagctg?gtctgcgtgt?t????????????????????????????????????21

<210>SEQ?ID?NO?324

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 324

caggagaacc?tgaatactcc?cag??????????????????????????????????23

<210>SEQ?ID?NO?325

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 325

aagcaccgga?ttcagttttg??????????????????????????????????????20

<210>SEQ?ID?NO?326

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 326

gtgtgaaagc?tgttttggcc??????????????????????????????????????20

<210>SEQ?ID?NO?327

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 327

ttatctccag?gcagtgggat?ggtt?????????????????????????????????24

<210>SEQ?ID?NO?328

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 328

cctgaagaac?tgtctcctgg?aat?????????????????????????????????23

<210>SEQ?ID?NO?329

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 329

tcttacttta?ccagaggtgg?ttgg????????????????????????????????24

<210>SEQ?ID?NO?330

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_004136

＜400〉sequence: 330

tttgctgcta?tgagggaggc?ag??????????????????????????????????22

<210>SEQ?ID?NO?331

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 331

ccccaactga?ggacattcag????????????????????????????????????20

<210>SEQ?ID?NO?332

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 332

ttgccactga?ctgtccaga????????????????????????????????????19

<210>SEQ?ID?NO?333

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 333

accttctatg?ctgccgttag?ct????????????????????????????????22

<210>SEQ?ID?NO?334

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 334

gtccttgtgg?gaaaagtacc?ac????????????????????????????????22

<210>SEQ?ID?NO?335

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 335

ccttctatgc?tgccgttagc??????????????????????????????????20

<210>SEQ?ID?NO?336

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 336

ctcagaatgg?gacaatccag?t????????????????????????????????21

<210>SEQ?ID?NO?337

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 337

aagcgacctt?gttcttcctt??????????????????????????????????20

<210>SEQ?ID?NO?338

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 338

ccttgttctt?cctttccttc?c????????????????????????????????21

<210>SEQ?ID?NO?339

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 339

ttgttcttcc?tttccttccg?ag???????????????????????????????22

<210>SEQ?ID?NO?340

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 340

attgccactg?actgtccaga?ag????????????????????????????????????22

<210>SEQ?ID?NO?341

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 341

tctcggaagg?aaaggaagaa??????????????????????????????????????20

<210>SEQ?ID?NO?342

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 342

cttcaataag?gcggcaactt??????????????????????????????????????20

<210>SEQ?ID?NO?343

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 343

ctgccttgta?tcacactctg?ct???????????????????????????????????22

<210>SEQ?ID?NO?344

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 344

ttctggacag?tcagtggcaa?t????????????????????????????????????21

<210>SEQ?ID?NO?345

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 345

gccttgtatc?acactctgct?tttc????????????????????????????????24

<210>SEQ?ID?NO?346

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 346

cctttggtga?aacgataggg?at??????????????????????????????????22

<210>SEQ?ID?NO?347

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 347

actggattgt?cccattctga?g???????????????????????????????????21

<210>SEQ?ID?NO?348

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 348

ggcaaatact?ggattgtccc?a????????????????????????????????????21

<210>SEQ?ID?NO?349

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 349

ggccactgct?tacatcaaca?g???????????????????????????????????21

<210>SEQ?ID?NO?350

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_014381

＜400〉sequence: 350

cttcaataag?gcggcaactt?????????????????????????????????????20

<210>SEQ?ID?NO?351

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 351

aaagacctga?caactgtggc?tgtg????????????????????????????????24

<210>SEQ?ID?NO?352

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 352

taatgatggc?ctgagcttac?agg????????????????????????????????23

<210>SEQ?ID?NO?353

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 353

acttcgcaaa?atggcccagg????????????????????????????????????20

<210>SEQ?ID?NO?354

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 354

gccaatgaac?ttcggagagg????????????????????????????????????20

<210>SEQ?ID?NO?355

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 355

acttcgcaaa?atggcccagg????????????????????????????????????20

<210>SEQ?ID?NO?356

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 356

gctgttgatg?taagcagtgg?c??????????????????????????????????21

<210>SEQ?ID?NO?357

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 357

tcgagcagag?aggactgtga?tga????????????????????????????23

<210>SEQ?ID?NO?358

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 358

tcgagcagag?aggactgtga?tga????????????????????????????23

<210>SEQ?ID?NO?359

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 359

tcgagcagag?aggactgtga?tga????????????????????????????23

<210>SEQ?ID?NO?360

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_014381

＜400〉sequence: 360

tgatggcctg?agcttacagg???????????????????????????????20

<210>SEQ?ID?NO?361

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 361

gttccagagc?aatcacatgg???????????????????????????????20

<210>SEQ?ID?NO?362

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 362

gaatatccct?cagcctggtg??????????????????????????????20

<210>SEQ?ID?NO?363

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 363

gaatatccct?cagcctggtg??????????????????????????????20

<210>SEQ?ID?NO?364

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 364

gaatatccct?cagcctggtg?????????????????????????????20

<210>SEQ?ID?NO?365

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 365

cggttccaga?gcaatcaca????????????????????????????19

<210>SEQ?ID?NO?366

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 366

ttccagagca?atcacatgg????????????????????????????19

<210>SEQ?ID?NO?367

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 367

agcctggtga?gggtgatgtt???????????????????????????20

<210>SEQ?ID?NO?368

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 368

agcctggtga?gggtgatgtt???????????????????????????20

<210>SEQ?ID?NO?369

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 369

agcctggtga?gggtgatgtt???????????????????????????20

<210>SEQ?ID?NO?370

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 370

agcctggtga?gggtgatgtt??????????????????????????20

<210>SEQ?ID?NO?371

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 371

cctggcatga?agatgagatc??????????????????????????20

<210>SEQ?ID?NO?372

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 372

tcagataggc?atcatagtag?ccct?????????????????????24

<210>SEQ?ID?NO?373

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 373

cagtcggttg?ccatgtttgt????????????????????????????20

<210>SEQ?ID?NO?374

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 374

agggcttcac?ttcctgatgc????????????????????????????20

<210>SEQ?ID?NO?375

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 375

cctggcatga?agatgagatc?a??????????????????????????21

<210>SEQ?ID?NO?376

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 376

cctggcatga?agatgagatc????????????????????????????20

<210>SEQ?ID?NO?377

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 377

agtcggttgc?catgtttgt?????????????????????????????19

<210>SEQ?ID?NO?378

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 378

agataggcat?catagtagcc?cttg???????????????????????24

<210>SEQ?ID?NO?379

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 379

catcatagta?gcccttgccc???????????????????????????20

<210>SEQ?ID?NO?380

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006441

＜400〉sequence: 380

cagtcggttg?ccatgtttg????????????????????????????19

<210>SEQ?ID?NO?381

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 381

cccaaaacat?cctggaatat?cc????????????????????????22

<210>SEQ?ID?NO?382

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 382

tgacaaacat?ggcaaccgac????????????????????????????????20

<210>SEQ?ID?NO?383

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 383

cttgatctca?tcttcatgcc?agg????????????????????????????23

<210>SEQ?ID?NO?384

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 384

gacaaacatg?gcaaccgact?gg?????????????????????????????22

<210>SEQ?ID?NO?385

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 385

cccaaaacat?cctggaatat?cc?????????????????????????????22

<210>SEQ?ID?NO?386

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 386

cccaaaacat?cctggaatat?cc?????????????????????????????22

<210>SEQ?ID?NO?387

＜211〉length: 25

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 387

cttgatctca?tcttcatgcc?aggcc??????????????????????????25

<210>SEQ?ID?NO?388

＜211〉length: 25

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 388

cttgatctca?tcttcatgcc?aggcc??????????????????????????25

<210>SEQ?ID?NO?389

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 389

acaaacatgg?caaccgactg?gg?????????????????????????????22

<210>SEQ?ID?NO?390

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006441

＜400〉sequence: 390

ttgatctcat?cttcatgcca?ggcc????????????????????????????24

<210>SEQ?ID?NO?391

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 391

tacagggaat?ggaacgacat?g??????????????????????????????21

<210>SEQ?ID?NO?392

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 392

tgaccctaaa?acagacccac?tt?????????????????????????????22

<210>SEQ?ID?NO?393

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 393

tagccaaaac?atcagccaag????????????????????????????????20

<210>SEQ?ID?NO?394

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 394

gtaccactgt?gagtgcagag?atg????????????????????????????23

<210>SEQ?ID?NO?395

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 395

caccaagtgg?agaatcgtgt???????????????????????????????20

<210>SEQ?ID?NO?396

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 396

tgtgcagaaa?atcatggagc???????????????????????????????20

<210>SEQ?ID?NO?397

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 397

atgacaggtg?ctctgtgtgc??????????????????????????????20

<210>SEQ?ID?NO?398

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 398

atgacaagtg?gcacaagctc????????????????????????20

<210>SEQ?ID?NO?399

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 399

tggtcagatt?tgggtgttgg???????????????????????20

<210>SEQ?ID?NO?400

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 400

gaaccaccta?ccgagaattt?g?????????????????????21

<210>SEQ?ID?NO?401

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 401

gcacgactgt?cacattgaac?a?????????????????????21

<210>SEQ?ID?NO?402

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 402

ccacttgtca?tcagccaaa????????????????????????19

<210>SEQ?ID?NO?403

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 403

tatccaggac?tcaaattctc?gg????????????????????22

<210>SEQ?ID?NO?404

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 404

ttctttccat?gaggacatcg???????????????????????20

<210>SEQ?ID?NO?405

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 405

agtcccctgt?gcaattcttt???????????????????????20

<210>SEQ?ID?NO?406

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 406

gcagttctta?cagccgtcta?tcc???????????????????23

<210>SEQ?ID?NO?407

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 407

gcactgacta?ctaagccttg?ggt????????????????????????????23

<210>SEQ?ID?NO?408

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 408

cctagaggca?agtctgtgga???????????????????????????????20

<210>SEQ?ID?NO?409

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 409

actactaagc?cttgggtcac?attc?????????????????????????24

<210>SEQ?ID?NO?410

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: 006159

＜400〉sequence: 410

taagtgggca?gtcaggattt?g????????????????????????????21

<210>SEQ?ID?NO?411

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 411

gcctgtattg?ccgctaatgt?gtgt?????????????????????????24

<210>SEQ?ID?NO?412

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 412

ggccatcgga?atgaagtcag?a????????????????????????????21

<210>SEQ?ID?NO?413

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 413

caccatgaag?ggaaccacct?????????????????????????????20

<210>SEQ?ID?NO?414

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 414

accaagtgga?gaatcgtgtg?aa??????????????????????????22

<210>SEQ?ID?NO?415

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 415

tattgatgag?tgtgggaccg?gga????????????????????????????????23

<210>SEQ?ID?NO?416

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 416

caccatgaag?ggaaccacct???????????????????????????????????20

<210>SEQ?ID?NO?417

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 417

ggatggtctg?tgactgtgag?aatc??????????????????????????????24

<210>SEQ?ID?NO?418

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 418

agccatcagt?gcttcccatt?t????????????????????????????????21

<210>SEQ?ID?NO?419

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 419

atggtctgtg?actgtgagaa?tcc?????????????????????????????23

<210>SEQ?ID?NO?420

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: 006159

＜400〉sequence: 420

ggctgtaaga?actgcacatg?cc??????????????????????????????22

<210>SEQ?ID?NO?421

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 421

ggctaaacac?tatcatgcca?ag??????????????????????????????22

<210>SEQ?ID?NO?422

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 422

ggctaaacac?tatcatgcca?ag??????????????????????????????22

<210>SEQ?ID?NO?423

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 423

ggctaaacac?tatcatgcca?ag????????????????????????????????22

<210>SEQ?ID?NO?424

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 424

ggctaaacac?tatcatgcca?ag???????????????????????????????22

<210>SEQ?ID?NO?425

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 425

ggctaaacac?tatcatgcca?ag???????????????????????????????22

<210>SEQ?ID?NO?426

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 426

ggctaaacac?tatcatgcca?ag??????????????????????????????22

<210>SEQ?ID?NO?427

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 427

ggctaaacac?tatcatgcca?ag?????????????????????????????22

<210>SEQ?ID?NO?428

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 428

ggctaaacac?tatcatgcca?ag?????????????????????????????22

<210>SEQ?ID?NO?429

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 429

ggctaaacac?tatcatgcca?ag?????????????????????????????22

<210>SEQ?ID?NO?430

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 430

ggctaaacac?tatcatgcca?ag?????????????????????????????22

<210>SEQ?ID?NO?431

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 431

caaactcctt?ctctcgttgc?tg????????????????????????????22

<210>SEQ?ID?NO?432

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 432

ttgctgctcc?ctttccaa????????????????????????????18

<210>SEQ?ID?NO?433

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 433

ctgctccctt?tccaactctt??????????????????????????20

<210>SEQ?ID?NO?434

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 434

ttgctgctcc?ctttccaac???????????????????????????19

<210>SEQ?ID?NO?435

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 435

actccttctc?tcgttgctgc??????????????????????????20

<210>SEQ?ID?NO?436

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 436

tccttctctc?gttgctgct??????????????????????????19

<210>SEQ?ID?NO?437

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 437

ttcaaactcc?ttctctcgtt?gc??????????????????????22

<210>SEQ?ID?NO?438

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 438

tgctgctccc?tttccaact?????????????????????????19

<210>SEQ?ID?NO?439

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 439

gttgctgctc?cctttccaa?????????????????????????19

<210>SEQ?ID?NO?440

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_012388

＜400〉sequence: 440

tcaaactcct?tctctcgttg?c????????????????????????????????????21

<210>SEQ?ID?NO?441

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 441

tgcagcagaa?gaggcaaaaa?g????????????????????????????????????21

<210>SEQ?ID?NO?442

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 442

tgcagcagaa?gaggcaaaaa?gaag????????????????????????????????24

<210>SEQ?ID?NO?443

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 443

tgcagcagaa?gaggcaaaaa????????????????????????????????????20

<210>SEQ?ID?NO?444

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 444

tgcagcagaa?gaggcaaaaa?gaag???????????????????????????????24

<210>SEQ?ID?NO?445

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 445

tgcagcagaa?gaggcaaaaa?g??????????????????????????????????21

<210>SEQ?ID?NO?446

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 446

tgcagcagaa?gaggcaaaaa?g??????????????????????????????????21

<210>SEQ?ID?NO?447

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 447

gcagcagaag?aggcaaaaag?aag????????????????????????????????23

<210>SEQ?ID?NO?448

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 448

tgcagcagaa?gaggcaaaaa?ga????????????????????????????????22

<210>SEQ?ID?NO?449

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 449

cagcagaaga?ggcaaaaaga?ag???????????????????????????????22

<210>SEQ?ID?NO?450

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_012388

＜400〉sequence: 450

cagcagaaga?ggcaaaaaga?ag???????????????????????????????22

<210>SEQ?ID?NO?451

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 451

gacctcaaga?aagcaacgaa??????????????????????????????????20

<210>SEQ?ID?NO?452

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 452

gacctcaaga?aagcaacgaa??????????????????????????????????20

<210>SEQ?ID?NO?453

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 453

gacctcaaga?aagcaacgaa?????????????????????????????????20

<210>SEQ?ID?NO?454

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 454

gacctcaaga?aagcaacgaa????????????????????????????????20

<210>SEQ?ID?NO?455

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 455

agaaagcaac?gaaaggaacg????????????????????????????????20

<210>SEQ?ID?NO?456

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 456

gcgacagtgc?ctaagacaga????????????????????????????????20

<210>SEQ?ID?NO?457

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 457

aacgaaagga?acgcaagaac????????????????????????????????20

<210>SEQ?ID?NO?458

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 458

agaaagcaac?gaaaggaacg????????????????????????????????20

<210>SEQ?ID?NO?459

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 459

aacgaaagga?acgcaagaac????????????????????????????????20

<210>SEQ?ID?NO?460

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 460

acgaaaggaa?cgcaagaaca????????????????????????????????20

<210>SEQ?ID?NO?461

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 461

cattgcagca?cctttactcc?tt?????????????????????????????22

<210>SEQ?ID?NO?462

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 462

tgcagcacct?ttactccttc????????????????????????????????20

<210>SEQ?ID?NO?463

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 463

cattgcagca?cctttactcc?t??????????????????????????????21

<210>SEQ?ID?NO?464

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 464

cattgcagca?cctttactcc????????????????????????????????20

<210>SEQ?ID?NO?465

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 465

gccacagcta?acatcattgc????????????????????????????????????20

<210>SEQ?ID?NO?466

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 466

atcatgccaa?agccagttgt????????????????????????????????????20

<210>SEQ?ID?NO?467

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 467

gccacagcta?acatcattgc????????????????????????????????????20

<210>SEQ?ID?NO?468

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 468

ggccacagct?aacatcattg???????????????????????????????????20

<210>SEQ?ID?NO?469

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 469

ggccacagct?aacatcattg??????????????????????????????????20

<210>SEQ?ID?NO?470

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001026

＜400〉sequence: 470

gccacagcta?acatcattgc??????????????????????????????????20

<210>SEQ?ID?NO?471

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 471

agaaagtcag?ggggactgca?aag?????????????????????????????23

<210>SEQ?ID?NO?472

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 472

gaaagtcagg?gggactgcaa?ag??????????????????????????????22

<210>SEQ?ID?NO?473

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 473

aaagtcaggg?ggactgcaaa?????????????????????????20

<210>SEQ?ID?NO?474

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 474

caatgttggt?gctggcaaaa?????????????????????????20

<210>SEQ?ID?NO?475

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 475

aatgaagaaa?gtcaggggga?ct?????????????????????22

<210>SEQ?ID?NO?476

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 476

cagaactcat?tttggtggtg?g??????????????????????21

<210>SEQ?ID?NO?477

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 477

aatgaagaaa?gtcaggggga?ct??????????????????????22

<210>SEQ?ID?NO?478

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 478

aatgaagaaa?gtcaggggga?ct?????????????????????22

<210>SEQ?ID?NO?479

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 479

aatgaagaaa?gtcaggggga?ct?????????????????????22

<210>SEQ?ID?NO?480

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001026

＜400〉sequence: 480

aatgaagaaa?gtcaggggga?ct?????????????????????22

<210>SEQ?ID?NO?481

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 481

ctcattttgg?tggtggcaag????????????????????????20

<210>SEQ?ID?NO?482

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 482

caactggctt?tggcatgatt????????????????????????20

<210>SEQ?ID?NO?483

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 483

cattttggtg?gtggcaagac????????????????????????20

<210>SEQ?ID?NO?484

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 484

agacaactgg?ctttggcat????????????????????????19

<210>SEQ?ID?NO?485

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 485

cagaactcat?tttggtggtg?g?????????????????????21

<210>SEQ?ID?NO?486

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 486

gaatgaagaa?agtcaggggg??????????????????????20

<210>SEQ?ID?NO?487

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 487

ctcattttgg?tggtggca????????????????????????18

<210>SEQ?ID?NO?488

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 488

agaactcatt?ttggtggtgg?c????????????????????21

<210>SEQ?ID?NO?489

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 489

acaactggct?ttggcatga???????????????????????19

<210>SEQ?ID?NO?490

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 490

tttggtggtg?gcaagacaa????????????????????????????????????????19

<210>SEQ?ID?NO?491

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 491

tgttcttgcg?ttcctttcg???????????????????????????????????????19

<210>SEQ?ID?NO?492

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 492

ctttgcagtc?cccctgactt?????????????????????????????????????20

<210>SEQ?ID?NO?493

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 493

ctttgcagtc?cccctgactt?????????????????????????????????????20

<210>SEQ?ID?NO?494

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 494

agtccccctg?actttcttca?tt?????????????????????????????????22

<210>SEQ?ID?NO?495

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 495

gttcttgcgt?tcctttcgtt???????????????????????????????????20

<210>SEQ?ID?NO?496

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 496

cattgcagca?cctttactcc?tt????????????????????????????????22

<210>SEQ?ID?NO?497

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 497

gtccccctga?ctttcttcat?t?????????????????????????????????21

<210>SEQ?ID?NO?498

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 498

tctgttcttg?cgttcctttc????????????????????????????20

<210>SEQ?ID?NO?499

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 499

gtccccctga?ctttcttcat?t?????????????????????????21

<210>SEQ?ID?NO?500

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_033022

＜400〉sequence: 500

tttgcagtcc?ccctgacttt??????????????????????????20

<210>SEQ?ID?NO?501

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 501

caactggctt?tggcatgatt??????????????????????????20

<210>SEQ?ID?NO?502

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 502

acgaaaggaa?cgcaagaaca?g????????????????????????21

<210>SEQ?ID?NO?503

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 503

cgaaaggaac?gcaagaacag??????????????????????????20

<210>SEQ?ID?NO?504

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 504

acgaaaggaa?cgcaagaaca?????????????????????????20

<210>SEQ?ID?NO?505

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 505

gacaactggc?tttggcatga?tt??????????????????????22

<210>SEQ?ID?NO?506

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 506

caatgttggt?gctggcaaaa????????????????????????20

<210>SEQ?ID?NO?507

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 507

acaactggct?ttggcatgat????????????????????????????20

<210>SEQ?ID?NO?508

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 508

gacaactggc?tttggcatga?tt????????????????????????22

<210>SEQ?ID?NO?509

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 509

acgaaaggaa?cgcaagaaca??????????????????????????20

<210>SEQ?ID?NO?510

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_033022

＜400〉sequence: 510

cgaaaggaac?gcaagaacag??????????????????????????20

<210>SEQ?ID?NO?511

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 511

atgccgtgga?caagagatg???????????????????????????19

<210>SEQ?ID?NO?512

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 512

tgtatgccgt?ggacaagaga?t????????????????????????21

<210>SEQ?ID?NO?513

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 513

ctgaactcaa?cgccatgctt??????????????????????????20

<210>SEQ?ID?NO?514

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 514

gactgagaag?gatttgctgc?t????????????????????????21

<210>SEQ?ID?NO?515

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 515

actgtatgcc?gtggacaaga????????????????????????????20

<210>SEQ?ID?NO?516

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 516

gaggtgaagc?atattgcctg????????????????????????????20

<210>SEQ?ID?NO?517

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 517

cagcaccaag?gacaggaaga?t??????????????????????????21

<210>SEQ?ID?NO?518

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 518

gactgtatgc?cgtggacaag?a??????????????????????????21

<210>SEQ?ID?NO?519

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 519

gtgaagcata?ttgcctggct????????????????????????????20

<210>SEQ?ID?NO?520

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 520

ccaaaacacc?agaacagca????????????????????????????19

<210>SEQ?ID?NO?521

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 521

gcctgtcctg?gtagcaaatg???????????????????????????20

<210>SEQ?ID?NO?522

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 522

cctgtcctgg?tagcaaatgt?aag???????????????????????23

<210>SEQ?ID?NO?523

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 523

tcacagccat?gaggacaggt????????????????????????????????????????20

<210>SEQ?ID?NO?524

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 524

atgtaaggtc?agatccaagg?ga?????????????????????????????????????22

<210>SEQ?ID?NO?525

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 525

aaggtcagat?ccaagggagg????????????????????????????????????????20

<210>SEQ?ID?NO?526

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 526

ctcttgtcca?cggcatacag?t??????????????????????????????????????21

<210>SEQ?ID?NO?527

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 527

atctttgcag?cgtaggatca????????????????????????????????????????20

<210>SEQ?ID?NO?528

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 528

gagcctgtcc?tggtagcaaa?t??????????????????????????????????????21

<210>SEQ?ID?NO?529

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 529

tctcttgtcc?acggcataca?g?????????????????????????????????????21

<210>SEQ?ID?NO?530

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_006750

＜400〉sequence: 530

gcagcgtagg?atcaacgtgt??????????????????????????????????????20

<210>SEQ?ID?NO?531

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 531

atgccacagc?tacccacttg?ttgc?????????????????????????????????24

<210>SEQ?ID?NO?532

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 532

tgccacagct?acccacttgt?tg????????????????????????????????22

<210>SEQ?ID?NO?533

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 533

gtgaagcata?ttgcctggct???????????????????????????????????20

<210>SEQ?ID?NO?534

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 534

catgccacag?ctacccactt?gt????????????????????????????????22

<210>SEQ?ID?NO?535

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 535

catgccacag?ctacccactt??????????????????????????????????20

<210>SEQ?ID?NO?536

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 536

gatggtggaa?gacagcaatg?gag??????????????????????????????23

<210>SEQ?ID?NO?537

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 537

cctctcaaaa?tgtgctttgc?tg???????????????????????????????22

<210>SEQ?ID?NO?538

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 538

tgccacagct?acccacttgt??????????????????????????????????20

<210>SEQ?ID?NO?539

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 539

atggtggaag?acagcaatgg?a????????????????????????????????21

<210>SEQ?ID?NO?540

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_006750

＜400〉sequence: 540

ccctctcaaa?atgtgctttg?ctgc????????????????????????????????24

<210>SEQ?ID?NO?541

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 541

acagctaccc?acttgttgcc????????????????????????????????????20

<210>SEQ?ID?NO?542

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 542

tagtggcagt?gaggactctg?gt????????????????????????????????22

<210>SEQ?ID?NO?543

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 543

atgtgctttg?ctgctagaaa?cc????????????????????????????????22

<210>SEQ?ID?NO?544

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 544

actgtatgcc?gtggacaaga?g????????????????????????????????21

<210>SEQ?ID?NO?545

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 545

caggatactt?gttcagggtt?gc???????????????????????????????22

<210>SEQ?ID?NO?546

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 546

ggatacttgt?tcagggttgc?c????????????????????????????????21

<210>SEQ?ID?NO?547

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 547

tctcttcagg?gtggagacac?a????????????????????????????????21

<210>SEQ?ID?NO?548

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 548

ggatacttgt?tcagggttgc?c????????????????????????????21

<210>SEQ?ID?NO?549

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 549

tctcttcagg?gtggagacac?a????????????????????????????21

<210>SEQ?ID?NO?550

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 550

tgactgtatg?ccgtggacaa?????????????????????????????20

<210>SEQ?ID?NO?551

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 551

ggatgacaga?tcccgatgt??????????????????????????????19

<210>SEQ?ID?NO?552

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 552

cattgctgtc?ttccaccatc??????????????????????????????20

<210>SEQ?ID?NO?553

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 553

catctcttgt?ccacggcata??????????????????????????????20

<210>SEQ?ID?NO?554

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 554

agagcctgtc?ctggtagcaa?a????????????????????????????21

<210>SEQ?ID?NO?555

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 555

ttcccttgag?atggtgaacc??????????????????????????????20

<210>SEQ?ID?NO?556

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 556

gcctccattt?tcccttga????????????????????????????????18

<210>SEQ?ID?NO?557

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 557

ccttgagatg?gtgaacccat?t????????????????????????????????21

<210>SEQ?ID?NO?558

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 558

agcctccatt?ttcccttgag?????????????????????????????????20

<210>SEQ?ID?NO?559

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 559

tgaatagtaa?gcctcacctc?ttgg????????????????????????????24

<210>SEQ?ID?NO?560

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_130845

＜400〉sequence: 560

ctgtcctggt?agcaaatgta?agg?????????????????????????????23

<210>SEQ?ID?NO?561

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 561

atttgctacc?aggacaggct?c??????????????????????????????21

<210>SEQ?ID?NO?562

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 562

tccctctcaa?aatgtgcttt?gc?????????????????????????????22

<210>SEQ?ID?NO?563

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 563

tgactgagaa?ggatttgctg?c??????????????????????????????21

<210>SEQ?ID?NO?564

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 564

tgccacagct?acccacttgt????????????????????????????????20

<210>SEQ?ID?NO?565

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 565

ccaagaggtg?aggcttacta?ttca????????????????????????????????24

<210>SEQ?ID?NO?566

＜211〉length: 25

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 566

ccaagaggtg?aggcttacta?ttcac???????????????????????????????25

<210>SEQ?ID?NO?567

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 567

caggatactt?gttcagggtt?gcc????????????????????????????????23

<210>SEQ?ID?NO?568

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 568

ccaagaggtg?aggcttacta?ttca???????????????????????????????24

<210>SEQ?ID?NO?569

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 569

aggatacttg?ttcagggttg?cca????????????????????????????????23

<210>SEQ?ID?NO?570

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_130845

＜400〉sequence: 570

tgccacagct?acccacttgt?tg?????????????????????????????????22

<210>SEQ?ID?NO?571

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 571

ctgggttatg?aagtgatgga?ag?????????????????????????????????22

<210>SEQ?ID?NO?572

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 572

ctgggttatg?aagtgatgga?ag?????????????????????????????????22

<210>SEQ?ID?NO?573

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 573

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?574

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 574

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?575

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 575

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?576

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 576

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?577

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 577

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?578

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 578

ctgggttatg?aagtgatgga?ag????????????????????????????22

<210>SEQ?ID?NO?579

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 579

agccaaaaga?agatggcaac???????????????????????????????20

<210>SEQ?ID?NO?580

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 580

ttggcagtgt?ctcaacaagc???????????????????????????????20

<210>SEQ?ID?NO?581

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 581

aaccagcgtt?ttggagga????????????????????????????????18

<210>SEQ?ID?NO?582

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 582

aaaccagcgt?tttggagg????????????????????????????????????18

<210>SEQ?ID?NO?583

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 583

accagcgttt?tggaggaag???????????????????????????????????19

<210>SEQ?ID?NO?584

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 584

aaaccagcgt?tttggaggaa??????????????????????????????????20

<210>SEQ?ID?NO?585

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 585

aaccagcgtt?ttggaggaag??????????????????????????????????20

<210>SEQ?ID?NO?586

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 586

aaaccagcgt?tttggagga???????????????????????????????????19

<210>SEQ?ID?NO?587

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 587

aaccagcgtt?ttggaggaa???????????????????????????????????19

<210>SEQ?ID?NO?588

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 588

accagcgttt?tggaggaa????????????????????????????????????18

<210>SEQ?ID?NO?589

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 589

ttgagcttgt?tgagacactg?c????????????????????????????????21

<210>SEQ?ID?NO?590

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_022133

＜400〉sequence: 590

atgaggggac?tgctacagac?a????????????????????????21

<210>SEQ?ID?NO?591

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 591

gtttccaggt?tttcgactag?ca???????????????????????22

<210>SEQ?ID?NO?592

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 592

gtttccaggt?tttcgactag?ca???????????????????????22

<210>SEQ?ID?NO?593

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 593

tgtttccagg?ttttcgacta?gc???????????????????????22

<210>SEQ?ID?NO?594

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 594

tgtttccagg?ttttcgacta?gc???????????????????????22

<210>SEQ?ID?NO?595

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 595

tgtttccagg?ttttcgacta?gc???????????????????????22

<210>SEQ?ID?NO?596

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 596

gtttccaggt?tttcgactag?ca???????????????????????22

<210>SEQ?ID?NO?597

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 597

gtttccaggt?tttcgactag?ca???????????????????????22

<210>SEQ?ID?NO?598

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 598

gtttccaggt?tttcgactag?ca????????????????????????22

<210>SEQ?ID?NO?599

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 599

ccataggaaa?ctctgcttcc?agt???????????????????????23

<210>SEQ?ID?NO?600

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_022133

＜400〉sequence: 600

agggccagtt?agaagactca?aa????????????????????????22

<210>SEQ?ID?NO?601

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 601

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?602

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 602

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?603

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 603

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?604

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 604

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?605

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 605

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?606

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 606

ctgggttatg?aagtgatgga?ag????????????????????????22

<210>SEQ?ID?NO?607

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 607

ctgggttatg?aagtgatgga?ag?????????????????????????????????22

<210>SEQ?ID?NO?608

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 608

ctgggttatg?aagtgatgga?ag?????????????????????????????????22

<210>SEQ?ID?NO?609

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 609

ttggcagtgt?ctcaacaagc????????????????????????????????????20

<210>SEQ?ID?NO?610

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 610

cccagaagaa?agctgggtag????????????????????????????????????20

<210>SEQ?ID?NO?611

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 611

aaaccagcgt?tttggagg??????????????????????????????????????18

<210>SEQ?ID?NO?612

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 612

aaccagcgtt?ttggaggaa?????????????????????????????????????19

<210>SEQ?ID?NO?613

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 613

accagcgttt?tggaggaa??????????????????????????????????????18

<210>SEQ?ID?NO?614

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 614

aaaccagcgt?tttggaggaa????????????????????????????????????20

<210>SEQ?ID?NO?615

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 615

aaaccagcgt?tttggagga????????????????????????????????????????19

<210>SEQ?ID?NO?616

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 616

aaccagcgtt?ttggaggaag??????????????????????????????????????20

<210>SEQ?ID?NO?617

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 617

aaccagcgtt?ttggagga???????????????????????????????????????18

<210>SEQ?ID?NO?618

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 618

accagcgttt?tggaggaag??????????????????????????????????????19

<210>SEQ?ID?NO?619

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 619

atgaggggac?tgctacagac?a???????????????????????????????????21

<210>SEQ?ID?NO?620

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152836

＜400〉sequence: 620

gcgttttgga?ggaagtgcta?????????????????????????????????????20

<210>SEQ?ID?NO?621

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 621

tgtttccagg?ttttcgacta?gc??????????????????????????????????22

<210>SEQ?ID?NO?622

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 622

tgtttccagg?ttttcgacta?gc??????????????????????????????????22

<210>SEQ?ID?NO?623

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 623

gtttccaggt?tttcgactag?ca????????????????????????????22

<210>SEQ?ID?NO?624

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 624

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?625

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 625

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?626

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 626

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?627

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 627

gtttccaggt?tttcgactag?ca????????????????????????????22

<210>SEQ?ID?NO?628

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 628

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?629

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 629

agggccagtt?agaagactca?aa????????????????????????????22

<210>SEQ?ID?NO?630

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152836

＜400〉sequence: 630

gagatgtttc?caggttttcg?ac????????????????????????????22

<210>SEQ?ID?NO?631

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 631

cggaaacagt?gaattgggaa?g?????????????????????????????21

<210>SEQ?ID?NO?632

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 632

gacactgaag?aacaaaatcc?gg????????????????????????????????????22

<210>SEQ?ID?NO?633

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 633

cggaaacagt?gaattgggaa?g?????????????????????????????????????21

<210>SEQ?ID?NO?634

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 634

cggaaacagt?gaattgggaa?g????????????????????????????????????21

<210>SEQ?ID?NO?635

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 635

cggaaacagt?gaattgggaa?g????????????????????????????????????21

<210>SEQ?ID?NO?636

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 636

cggaaacagt?gaattgggaa?g????????????????????????????????????21

<210>SEQ?ID?NO?637

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 637

cactgaagaa?caaaatccgg?????????????????????????????????????20

<210>SEQ?ID?NO?638

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 638

gacactgaag?aacaaaatcc?gg??????????????????????????????????22

<210>SEQ?ID?NO?639

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 639

cggaaacagt?gaattgggaa?g???????????????????????????????????21

<210>SEQ?ID?NO?640

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 640

gacactgaag?aacaaaatcc?gg????????????????????????????????????22

<210>SEQ?ID?NO?641

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 641

ttaaaccagc?gttttggagg???????????????????????????????????????20

<210>SEQ?ID?NO?642

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 642

ttaaaccagc?gttttggagg???????????????????????????????????????20

<210>SEQ?ID?NO?643

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 643

taaaccagcg?ttttggagga????????????????????????????????????????20

<210>SEQ?ID?NO?644

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 644

taaaccagcg?ttttggagg?????????????????????????????????????????19

<210>SEQ?ID?NO?645

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 645

aaccagcgtt?ttggaggaag????????????????????????????????????????20

<210>SEQ?ID?NO?646

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 646

aaaccagcgt?tttggaggaa????????????????????????????????????????20

<210>SEQ?ID?NO?647

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 647

aaaccagcgt?tttggagg??????????????????????????????????????????18

<210>SEQ?ID?NO?648

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 648

aaaccagcgt?tttggagg????????????????????????????????????18

<210>SEQ?ID?NO?649

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 649

accagcgttt?tggaggaag???????????????????????????????????19

<210>SEQ?ID?NO?650

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_152837

＜400〉sequence: 650

taaaccagcg?ttttggagga?????????????????????????????????20

<210>SEQ?ID?NO?651

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 651

tgtttccagg?ttttcgacta?gc?????????????????????????????22

<210>SEQ?ID?NO?652

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 652

gtttccaggt?tttcgactag?ca?????????????????????????????22

<210>SEQ?ID?NO?653

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 653

gtttccaggt?tttcgactag?ca????????????????????????????22

<210>SEQ?ID?NO?654

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 654

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?655

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 655

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?656

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 656

gtttccaggt?tttcgactag?ca???????????????????????????22

<210>SEQ?ID?NO?657

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 657

gtttccaggt?tttcgactag?ca????????????????????????????22

<210>SEQ?ID?NO?658

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 658

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?659

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 659

tgtttccagg?ttttcgacta?gc????????????????????????????22

<210>SEQ?ID?NO?660

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_152837

＜400〉sequence: 660

gtttccaggt?tttcgactag?ca???????????????????????????22

<210>SEQ?ID?NO?661

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 661

tgtgatcctt?gcataccgg???????????????????????????????19

<210>SEQ?ID?NO?662

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 662

agaaaggctg?ctcagtgtga??????????????????????????????20

<210>SEQ?ID?NO?663

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 663

gttccttgtt?ttcaccctgg??????????????????????????????20

<210>SEQ?ID?NO?664

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 664

agctgtcggc?actgtaactc?????????????????????????????20

<210>SEQ?ID?NO?665

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 665

ctgctcagtg?tgatccttgc????????????????????????????????20

<210>SEQ?ID?NO?666

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 666

agagctgtcg?gcactgtaac?t??????????????????????????????21

<210>SEQ?ID?NO?667

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 667

agaaaggctg?ctcagtgtga?t?????????????????????????????21

<210>SEQ?ID?NO?668

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 668

agcccaccca?cttaccttat?g?????????????????????????????21

<210>SEQ?ID?NO?669

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 669

cagcatagaa?aggctgctca?g????????????????????????????21

<210>SEQ?ID?NO?670

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 670

gctcagtgtg?atccttgca??????????????????????????????19

<210>SEQ?ID?NO?671

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 671

attggcagtg?atggtgcag??????????????????????????????19

<210>SEQ?ID?NO?672

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 672

attggcagtg?atggtgcag??????????????????????????????19

<210>SEQ?ID?NO?673

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 673

ccggttttcg?gtaatcctc????????????????????????????????19

<210>SEQ?ID?NO?674

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 674

tcagcgaagg?gtttggaa?????????????????????????????????18

<210>SEQ?ID?NO?675

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 675

tgatggtgca?gttgcgaa?????????????????????????????????18

<210>SEQ?ID?NO?676

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 676

cagcgaaggg?tttggaaga????????????????????????????????19

<210>SEQ?ID?NO?677

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 677

attggcagtg?atggtgca?????????????????????????????????18

<210>SEQ?ID?NO?678

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 678

agaagatcac?aaggatgcga???????????????????????????????20

<210>SEQ?ID?NO?679

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 679

attggcagtg?atggtgca?????????????????????????????????18

<210>SEQ?ID?NO?680

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_001242

＜400〉sequence: 680

tggcagtgat?ggtgcagtt????????????????????????????????19

<210>SEQ?ID?NO?681

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 681

agctgtcggc?actgtaactc?tg????????????????????????????22

<210>SEQ?ID?NO?682

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 682

agagctgtcg?gcactgtaac?tc????????????????????????????????22

<210>SEQ?ID?NO?683

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 683

cctgttcctc?catcaacgaa?g?????????????????????????????????21

<210>SEQ?ID?NO?684

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 684

caactgcacc?atcactgcca??????????????????????????????????20

<210>SEQ?ID?NO?685

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 685

agctgtcggc?actgtaactc?tg??????????????????????????????22

<210>SEQ?ID?NO?686

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 686

aactgcacca?tcactgccaa?tgc????????????????????????????23

<210>SEQ?ID?NO?687

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 687

agagctgtcg?gcactgtaac?tct?????????????????????????????23

<210>SEQ?ID?NO?688

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 688

aaagatccct?gtgcagctcc?gat?????????????????????????????23

<210>SEQ?ID?NO?689

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 689

agagctgtcg?gcactgtaac?tct????????????????????????????23

<210>SEQ?ID?NO?690

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_001242

＜400〉sequence: 690

agagctgtcg?gcactgtaac?tctg????????????????????????????????24

<210>SEQ?ID?NO?691

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 691

cctctcggtt?ccataaccat?a??????????????????????????????????21

<210>SEQ?ID?NO?692

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 692

ccagaagtta?tccagagtct?ccc????????????????????????????????23

<210>SEQ?ID?NO?693

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 693

agtgaaagca?gtccaacttg?c?????????????????????????????????21

<210>SEQ?ID?NO?694

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 694

cctttgagat?tggtgcatgg???????????????????????????????????20

<210>SEQ?ID?NO?695

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 695

tcagcagctc?gtcagaaaa????????????????????????????????????19

<210>SEQ?ID?NO?696

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 696

tgggagatgc?taacaaggga???????????????????????????????????20

<210>SEQ?ID?NO?697

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 697

cagtaacaga?agtgaggaga?tgg??????????????????????????????23

<210>SEQ?ID?NO?698

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 698

gcagtccaac?ttgccattc????????????????????????????????19

<210>SEQ?ID?NO?699

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 699

ccagagtctc?cctgtgtcag?aa????????????????????????????22

<210>SEQ?ID?NO?700

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 700

ctgggagatg?ctaacaaggg???????????????????????????????20

<210>SEQ?ID?NO?701

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 701

tcccttgtta?gcatctccca?g?????????????????????????????21

<210>SEQ?ID?NO?702

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 702

gcaactgctt?ggaatggtt????????????????????????????????19

<210>SEQ?ID?NO?703

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 703

ggaacgctgt?aaagaagtgt?tg????????????????????????????22

<210>SEQ?ID?NO?704

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 704

atgcccatgt?ctttcaggtc??????????????????????????????20

<210>SEQ?ID?NO?705

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 705

atgcccatgt?ctttcaggtc??????????????????????????????20

<210>SEQ?ID?NO?706

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 706

ttgcttgaat?gatggccg????????????????????????????????18

<210>SEQ?ID?NO?707

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 707

cgtttcttga?cttgaggtct?ctgt????????????????????????????????????24

<210>SEQ?ID?NO?708

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 708

ttgctctggg?aacgctgta?????????????????????????????????????????19

<210>SEQ?ID?NO?709

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 709

gcaactgctt?ggaatggtt?????????????????????????????????????????19

<210>SEQ?ID?NO?710

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_016653

＜400〉sequence: 710

tgcttgaatg?atggccgt??????????????????????????????????????????18

<210>SEQ?ID?NO?711

＜211〉length: 29

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 711

gaagttatcc?agagtctccc?tgtgtcaga??????????????????????????????29

<210>SEQ?ID?NO?712

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 712

tctctgggag?atgctaacaa?gg????????????????????????????????????22

<210>SEQ?ID?NO?713

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 713

tcagatggca?accctggaag??????????????????????????????????????20

<210>SEQ?ID?NO?714

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 714

acaacattac?agggaagcgg?c????????????????????????????????????21

<210>SEQ?ID?NO?715

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 715

acaacattac?agggaagcgg?c????????????????????????????????21

<210>SEQ?ID?NO?716

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 716

ctgttacatc?agtgttggga?agc??????????????????????????????23

<210>SEQ?ID?NO?717

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 717

tatgacctgg?gccactgatg??????????????????????????????????20

<210>SEQ?ID?NO?718

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 718

ttcagatggc?aaccctggaa?g????????????????????????????????21

<210>SEQ?ID?NO?719

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 719

tctctgggag?atgctaacaa?ggga????????????????????????????24

<210>SEQ?ID?NO?720

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_016653

＜400〉sequence: 720

ttgccccaga?agttttgctg?aac????????????????????????????23

<210>SEQ?ID?NO?721

＜211〉length: 18

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 721

tgccccagaa?gttttgct??????????????????????????????????18

<210>SEQ?ID?NO?722

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 722

tctcagcttt?aaggagcagg?a?????????????????????????????21

<210>SEQ?ID?NO?723

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 723

attcctacac?aacaaggcgg?a????????????????????????21

<210>SEQ?ID?NO?724

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 724

atgacacgag?ccttcctga???????????????????????????19

<210>SEQ?ID?NO?725

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 725

gagatgctaa?caagggaggt?cc???????????????????????22

<210>SEQ?ID?NO?726

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 726

tgttacatca?gtgttgggaa?gc???????????????????????22

<210>SEQ?ID?NO?727

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 727

catggatggc?tccagaagtt?at???????????????????????22

<210>SEQ?ID?NO?728

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 728

tgatctcagc?tttaaggagc?ag???????????????????????22

<210>SEQ?ID?NO?729

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 729

aaagctgaca?gagcagtcca?ac??????????????????????22

<210>SEQ?ID?NO?730

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 730

gttttgggag?tgtttatcga?gc??????????????????????22

<210>SEQ?ID?NO?731

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 731

aggaaggctc?gtgtcatttg?????????????????????????20

<210>SEQ?ID?NO?732

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 732

acatctctgc?actgtttgac?tcc????????????????????????????????23

<210>SEQ?ID?NO?733

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 733

tgctctgtca?gcttttgctc????????????????????????????????????20

<210>SEQ?ID?NO?734

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 734

ctgctcctta?aagctgagat?cac????????????????????????????????23

<210>SEQ?ID?NO?735

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 735

agcttcccaa?cactgatgta?acag???????????????????????????????24

<210>SEQ?ID?NO?736

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 736

gccttgttgt?gtaggaatga?gt?????????????????????????????????22

<210>SEQ?ID?NO?737

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 737

ttccactaca?agccaagcta?ct????????????????????????????????22

<210>SEQ?ID?NO?738

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 738

ctgcactgtt?tgactcctct?gt????????????????????????????????22

<210>SEQ?ID?NO?739

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 739

cagacttggg?ttcatgcca???????????????????????????????????19

<210>SEQ?ID?NO?740

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_133646

＜400〉sequence: 740

aatgccatag?ttgggaggtt????????????????????????????????20

<210>SEQ?ID?NO?741

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 741

acggccatca?ttcaagcaaa????????????????????????????????20

<210>SEQ?ID?NO?742

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 742

tgtgggagca?aaagctgaca????????????????????????????????20

<210>SEQ?ID?NO?743

＜211〉length: 27

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 743

cgtgatctca?gctttaagga?gcaggag????????????????????????27

<210>SEQ?ID?NO?744

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 744

actcattcct?acacaacaag?gcgg??????????????????????????24

<210>SEQ?ID?NO?745

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 745

ttgccccaga?agttttgctg???????????????????????????????20

<210>SEQ?ID?NO?746

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 746

cggccatcat?tcaagcaaat???????????????????????????????20

<210>SEQ?ID?NO?747

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 747

tctctgggag?atgctaacaa?ggg??????????????????????????23

<210>SEQ?ID?NO?748

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 748

tgtgggagca?aaagctgaca?gag????????????????????????????????????23

<210>SEQ?ID?NO?749

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 749

atcacagcaa?caagtaacgg?gg??????????????????????????????????????22

<210>SEQ?ID?NO?750

＜211〉length: 23

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_133646

＜400〉sequence: 750

caggacaagg?aggtggctgt?aaa?????????????????????????????????????23

<210>SEQ?ID?NO?751

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 751

aacaccgaag?aaagcgaaga????????????????????????????????????????20

<210>SEQ?ID?NO?752

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 752

caccgaagaa?agcgaagaag????????????????????????????????????????20

<210>SEQ?ID?NO?753

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 753

agcgtagcgg?agtttctctg????????????????????????????????????????20

<210>SEQ?ID?NO?754

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 754

agcgtagcgg?agtttctctg????????????????????????????????????????20

<210>SEQ?ID?NO?755

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 755

actctggtgc?aaacgaaagg????????????????????????????????????????20

<210>SEQ?ID?NO?756

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 756

aaacaccgaa?gaaagcgaag????????????????????????????????????????20

<210>SEQ?ID?NO?757

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 757

agccaagccc?aaggttaaaa????????????????????????????????????20

<210>SEQ?ID?NO?758

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 758

agccaagccc?aaggttaaaa????????????????????????????????????20

<210>SEQ?ID?NO?759

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 759

cactctggtg?caaacgaaag????????????????????????????????????20

<210>SEQ?ID?NO?760

＜211〉length: 19

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 760

gccaagccca?aggttaaaa????????????????????????????????????19

<210>SEQ?ID?NO?761

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 761

agccttagca?gcacttttgg????????????????????????????????????20

<210>SEQ?ID?NO?762

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 762

agccttagca?gcacttttgg????????????????????????????????????20

<210>SEQ?ID?NO?763

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 763

ctttcgtttg?caccagagtg????????????????????????????????????20

<210>SEQ?ID?NO?764

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 764

ccaggctctt?gagaccaagt????????????????????????????????????20

<210>SEQ?ID?NO?765

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 765

ccccaactgg?cttcttaggt????????????????????????????????????????????20

<210>SEQ?ID?NO?766

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 766

agccttagca?gcacttttgg????????????????????????????????????????????20

<210>SEQ?ID?NO?767

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 767

ttcttcgctt?tcttcggtgt???????????????????????????????????????????20

<210>SEQ?ID?NO?768

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 768

tcttcgcttt?cttcggtgtt???????????????????????????????????????????20

<210>SEQ?ID?NO?769

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 769

ccccaactgg?cttcttaggt???????????????????????????????????????????20

<210>SEQ?ID?NO?770

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: primer is used for registration number: NM_005319

＜400〉sequence: 770

ttcttcgctt?tcttcggtgt??????????????????????????????????????????20

<210>SEQ?ID?NO?771

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 771

gtaaccaaga?aagtggctaa?gagc?????????????????????????????????????24

<210>SEQ?ID?NO?772

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 772

gtaaccaaga?aagtggctaa?gagc????????????????????????????????????24

<210>SEQ?ID?NO?773

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 773

ggagaaaaac?aacagccgta?tc??????????????????????????????????????????22

<210>SEQ?ID?NO?774

＜211〉length: 22

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 774

ggagaaaaac?aacagccgta?tc??????????????????????????????????????????22

<210>SEQ?ID?NO?775

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 775

aagcccaagg?ttaaaaaggc?????????????????????????????????????????????20

<210>SEQ?ID?NO?776

＜211〉length: 24

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 776

gtaaccaaga?aagtggctaa?gagc????????????????????????????????????????24

<210>SEQ?ID?NO?777

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 777

aaacctaaga?agccagttgg?g???????????????????????????????????????????21

<210>SEQ?ID?NO?778

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 778

aaacctaaga?agccagttgg?g??????????????????????????????????????????21

<210>SEQ?ID?NO?779

＜211〉length: 20

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 779

aagcccaagg?ttaaaaaggc????????????????????????????????????????????20

<210>SEQ?ID?NO?780

＜211〉length: 21

＜212〉type: DNA

＜213〉biology: artificial sequence

＜220〉feature:

＜223〉out of Memory: probe is used for registration number: NM_005319

＜400〉sequence: 780

aaacctaaga?agccagttgg?g?????????????????????????????????????????21

Claims (32)

1. the bladder cancer or the method in bladder cancer stage in the screening test object, it comprises:

(a) determine the level of the RNA product of first biomarker in the separation blood sample of described object, described first biomarker is selected from the group of the biomarker of listing in table 13; With the level of the RNA product of second biomarker, described second biomarker is selected from the group of described biomarker; With

(b) for each of described first and second biomarkers, with described RNA product level with by described biomarker coding and separate at one or more contrast objects that the control level of detected RNA compares in the blood sample, wherein for each described biomarker, to statistical significant difference between the gentle described control level of the described RNA product water of step (a) really definiteness show bladder cancer or described bladder cancer stage in the described checked object.

2. the bladder cancer or the method in bladder cancer stage in the screening test object, it comprises:

(a) determine that in the separation blood sample of described object described biomarker is selected from the group of the biomarker of listing in table 13 by the RNA product level of biomarker coding; With

(b) described level and control level are compared, described control level is the level by described biomarker RNA product coding and that find in the separation blood sample of one or more contrast objects, wherein to difference between described level of step (a) and the described control level really definiteness show bladder cancer or described bladder cancer stage in the described checked object.

3. according to claim 1 or 2 each methods, the group of wherein said biomarker is limited to SNX16, CSPG6, IGFBP7, CTSD and TNFRSF7, NELL2 and CHD2.

4. according to claim 1 or 2 each methods, wherein said separation blood sample is made up of whole blood.

5. according to claim 1 or 2 each methods, wherein said separation blood sample is made up of drop of blood.

According to the process of claim 1 wherein by at first from the described blood sample of described checked object isolation of RNA come determining of implementation step (a) for the described of the described rna level of each described biomarker.

7. according to the process of claim 1 wherein that using real-time quantitative RT-PCR (QRT-PCR) comes the described of described rna level for each described biomarker of implementation step (a) to determine.

8. according to the process of claim 1 wherein by hybridizing to come the described of described rna level for each described biomarker of implementation step (a) to determine by the described RNA product of each described biomarker coding and the array that comprises with at least a probe of described RNA product complementary of each described biomarker.

According to the process of claim 1 wherein by will coming from described RNA product cDNA, PCR product or EST and comprise with the array of described cDNA, PCR product or at least a probe of EST complementary of each described biomarker and hybridize to come the described of described rna level for each described biomarker of implementation step (a) to determine.

10. according to the process of claim 1 wherein that described control level is to come from described one or more individuality that does not have bladder cancer.

11. the bladder cancer or the method in bladder cancer stage in the screening test object, it comprises:

(a) determine the level of the protein of first biomarker in the separation blood sample of described object, described first biomarker is selected from the group of the biomarker of listing in table 13; With the level of the protein of second biomarker, described second biomarker is selected from the group of described biomarker; With

(b) for each of described first and second biomarkers, with described protein level with by described biomarker coding and separate at one or more contrast objects that the control level of detected protein compares in the blood sample, wherein for each described biomarker, to statistical significant difference between described protein level of step (a) and the described control level really definiteness show bladder cancer or described bladder cancer stage in the described checked object.

12. the bladder cancer or the method in bladder cancer stage in the screening test object, it comprises:

(a) determine that in the separation blood sample of described object by biomarker encoded protein matter product level, described biomarker is selected from the group of the biomarker of listing in table 13; With

(b) described level and control level are compared, described control level is the level by described biomarker protein coding and that find in the separation blood sample of one or more contrast objects, wherein to difference between described level of step (a) and the described control level really definiteness show bladder cancer or described bladder cancer stage in the described checked object.

13. comprise the composition of the set of two or more isolated proteins, the protein of at least two kinds of unique biomarkers of described isolated protein selective binding, wherein each described unique biomarker is selected from the group of the listed biomarker of table 13.

14. comprise the composition of the set of two or more groups biomarker Auele Specific Primer, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, wherein each described unique biomarker is selected from the group of the listed biomarker of table 13.

15. comprise the composition of the set of three groups or more groups biomarker Auele Specific Primers, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, and each described unique biomarker is selected from the group of the listed biomarker of table 13.

16. comprise the composition of the set of four groups or more groups biomarker Auele Specific Primers, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, and each described unique biomarker is selected from the group of the listed biomarker of table 13.

17. comprise the composition of the set of five groups or more groups biomarker Auele Specific Primers, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, and each described unique biomarker is selected from the group of the listed biomarker of table 13.

18. comprise the composition of the set of two or more groups biomarker Auele Specific Primer, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, and each described unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, NELL2 and CHD2.

19. comprise the composition of the set of three groups or more groups biomarker Auele Specific Primers, wherein each group selection amplification and unique biomarker complementary double-stranded DNA, and each described unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, NELL2 and CHD2.

20. comprise the composition of the set of two or more isolated proteins, the protein of at least two kinds of unique biomarkers of described isolated protein selective binding, wherein each unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, NELL2 and CHD2.

21. the composition of claim 20, wherein said isolated protein is an antibody.

22. the purposes of the described composition of claim 13, it is used for the existence of selective binding at least two kinds of different proteins of isolating biological sample, wherein every kind of described protein is coded by unique biomarker, and every kind of described unique biomarker is selected from the listed biomarker of table 13.

23. the purposes of the described composition of claim 14, it is used for selective amplification at isolating biological sample and at least two kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the listed biomarker of table 13.

24. the purposes of the described composition of claim 15, it is used for selective amplification at isolating biological sample and at least three kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the listed biomarker of table 13.

25. the purposes of the described composition of claim 16, it is used for selective amplification at isolating biological sample and at least four kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the listed biomarker of table 13.

26. the purposes of the described composition of claim 17, it is used for selective amplification at isolating biological sample and at least five kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the listed biomarker of table 13.

27. the purposes of the described composition of claim 18, it is used for selective amplification at isolating biological sample and at least two kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, NELL2 and CHD2.

28. the purposes of the described composition of claim 19, it is used for selective amplification at isolating biological sample and at least three kinds of unique biomarker complementary double-stranded DNAs, and every kind of described unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7 and CTSD and TNFRSF7, NELL2 and CHD2.

29. the purposes of each described composition in the claim 13,14,15,16,17,18 or 19, it was used in the blood sample screening bladder cancer or the bladder cancer stage of separating the self-checking object.

30. be used to implement the test kit of the described method of claim 1, it comprises at least two group biomarker Auele Specific Primers, wherein every group of biomarker Auele Specific Primer produces and unique biomarker complementary double-stranded DNA, and every kind of unique biomarker is selected from described biomarker group;

(b) has the enzyme of reverse transcriptase activity;

(c) have the active enzyme of heat-stable DNA polymerase and

(d) marking tool;

Wherein use the quantitative expression level that each described primer sets detects unique biomarker described in the checked object.

31. be used to implement the test kit of the described method of claim 1, it comprises at least two group biomarker Auele Specific Primers, wherein every group of biomarker Auele Specific Primer produces and unique biomarker complementary double-stranded DNA, and every kind of unique biomarker is selected from the group of the biomarker of being made up of SNX16, CSPG6, IGFBP7, CTSD, TNFRSF7, NELL2 and CHD2;

(b) has the enzyme of reverse transcriptase activity;

(c) have the active enzyme of heat-stable DNA polymerase and

(d) marking tool;

Wherein use the quantitative expression level that each described primer sets detects unique biomarker described in the checked object.

32. a test kit, it comprises:

(a) each described composition in the claim 14,15,16,17,18 or 19;

(b) has the enzyme of reverse transcriptase activity;

(c) have the active enzyme of heat-stable DNA polymerase and

(d) marking tool;

Wherein use the quantitative expression level that each described biomarker Auele Specific Primer group detects unique biomarker described in the checked object.

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