CN1771337B - A plynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucle - Google Patents
A plynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucle Download PDFInfo
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Abstract
Provided is a polynucleotide including at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOS: 1-12 and including a nucleotide at position 101 of the nucleotide sequence, or a complementary polynucleotide thereof.
Description
Technical field
The present invention relates to and the Cancer-Related polynucleotide of colorectum, the microarray that comprises these polynucleotide and diagnostic kit, and the method for analysis and the Cancer-Related polynucleotide of colon.
Background technology
The genome of all organisms stands spontaneous mutation, produces the variant form (Gusella, Ann.Rev.Biochem.55,831-854 (1986)) of ancestors (progenitor) nucleotide sequence in the process that they are constantly evolved.Variant form can be given evolution advantage or the shortcoming with respect to ancestors' form, or can be equivalents.In some instances, variant form is given lethal shortcoming, and not hereditary offspring to this organism.In other instance, variant form is given species with the evolution advantage, and finally for good and all is integrated among most of members' the DNA of these species and becomes ancestors' form effectively.In many instances, ancestors and variant form are all survived and in the population of species, are coexisted.The coexistence of the various ways of sequence causes polymorphum.
In polymorphum, known several types comprises that restriction fragment length polymorphism (RFLP), short series connection repetition (STR), variable string join repetition (VNTR) and SNP (SNP).Wherein, SNP has the form of single nucleotide variations between the individuality of same species.When SNP took place in albumen coded sequence, some polymorphum forms can cause that amino acid whose non-synonym changes, and cause giving expression to albumen defective or variation.On the other hand, when SNP in non--encoding sequence, when for example in intron, taking place, the splice variant of some caused mRNA of these polymorphums, this also cause defective or the variation proteic expression.Other SNP can not have phenotypic effect (phenotypic effect) fully.
According to estimates, people SNP is with per 1, and 1 frequency takes place among the 000bp.When such SNP influences the expression of phenotype, during like disease, the polynucleotide that contain this SNP can be used as primer or the probe of diagnosing this disease.Also can be used for the diagnosis of this disease specifically with this SNP bonded monoclonal antibody.At present, the research of the nucleotide sequence of SNP and function is just undertaken by many institutes.The nucleotide sequence of the people SNP that identifies has been processed DB to make it easy to acquisition with other experimental results.
Though discovery so far shows that concrete SNP is present on people's gene group or the cDNAs, does not also disclose the phenotypic effect of SNP.Remove outside a spot of SNP, the function of most of SNP is yet not open.
Colorectal carcinoma is a kind ofly to comprise the cancer that Korea S is very common at world wide.In Korea S, colorectal carcinoma all is the 4th common cancer in masculinity and femininity.In the middle of the death that is caused by cancer, colorectal carcinoma is number four, and for causing the dead reason of seven people in per 100,000 populations.In in the past 10 years, the mortality ratio of colorectal carcinoma increases about 80%.
The generation of known colorectal carcinoma is mainly caused by environmental factors.The quick westization of diet is the principal element that colorectal carcinoma takes place with animal tallow or proteic excess ingestion.Yet known about 5% colorectal carcinoma case is caused by the reason of gene.
Colorectal carcinoma patient more than 90% is the people who surpassed 40 years old at those ages.The generation of known colorectal carcinoma; Removing the age surpasses outside 40 years old the people, in the crowd's (high-risk group) who has with colorectal carcinoma, inflammatory bowel (inflammatory bowel disease), polyp of colon (colonic polyp), ovarian cancer (ovarian cancer), family history that uterus carcinoma (uterine cancer) is relevant with breast cancer (breast cancer), is more frequent.The generation of colorectal carcinoma in the 30-40 youngster in year then mainly is that the reason of gene is preponderated.
The early detection of colorectal carcinoma is guaranteed almost 100% curative ratio.Yet because colorectal carcinoma does not have specific early symptom, early detection is difficult usually.Fecal occult blood (fecal occult blood) check (being used for detecting the blood of stool trace) is used as screening test usually to detect colorectal carcinoma, especially when described cancer does not cause any symptom.The fecal occult blood check is to select to need further the accurately method of the individuality of inspection.Yet, because this inspection has the vacation-positive findings and the vacation-negative findings of height ratio, thus it and be not suitable for early diagnosis.At present, the accurate diagnosis of colorectal carcinoma is accomplished through barium enema inspection (barium enema examination), splanchnoscopy (endoscopy), actinoscopy (radiation examination) or the like.The common result of treatment that adopts the tumor marker (tumor marker) that is known as CEA (CEACAMS (carcinoembryonic antigen)) to detect the progress stage of colorectal carcinoma and assess colorectal carcinoma.But, do not generally acknowledge generally that also also attested tumor marker can come early diagnosis or prediction colorectal carcinoma through blood count.Though reported that several kinds of affinity tags are used for commute and suffer from high-risk group of patient of colorectal carcinoma and screen or early diagnosis, these affinity tags have limitation when being used for patient that great majority suffer from colorectal carcinoma.
Various cancers and concurrent disease comprise colorectal carcinoma, and the most serious problem is in its early diagnosis or the prognosis, and when cancer and concurrent disease were in late period, this diagnosis or prediction can be carried out through physical method.Yet the preliminary completion of the development of recent various Protocols in Molecular Biologies and the Human Genome Project makes finds that direct/gene or genovariation indirect and disease-related becomes possibility.Therefore, use the early diagnosis of the generation of gene prediction disease, the conventional phenotype of replacement application-or phenotype disease-dependent diagnostic method, become feasible.At present, biological chemistry or molecular biology method can be used for the colorectum cancerous diagnose.Owing to lack the information of dependency between the gene relevant or genovariation and gene or genovariation and the colorectum cancer morbidity, do not adopt molecular biology method colorectal carcinoma to be met the early diagnosis of patient and doctor's demand at present with this cancer.In addition, in most of diagnosed SARS cases of using single creature affinity tag, the sensitivity of this affinity tag and specificity can not be simultaneously satisfactory usually.Normally, if highly sensitive, specificity is just low, and vice versa.Owing to this reason, the possibility that makes a mistake in the diagnosis is high, so just be difficult to the levels of accuracy that reaches required.Therefore, single creature is learned affinity tag and only is used as the diagnostic marker that preliminary screening is carried out in accurate inspection.
Detailed Description Of The Invention
Technical object of the present invention
The invention provides the polynucleotide that contain with the Cancer-Related SNP of colorectum.
The present invention also provides microarray and colorectal carcinoma diagnostic kit, its every kind polynucleotide that contain with the Cancer-Related SNP of colorectum.
The present invention also provides the method for application and the Cancer-Related polynucleotide diagnosis of colorectal cancer of colorectum.
Content of the present invention
The invention provides at least 10 contiguous nucleotides that comprise the nucleotide sequence that is selected from nucleotide sequence SEQ ID NOS:1-12, and comprise the polynucleotide of Nucleotide of the pleomorphism site (the 101st) of this nucleotide sequence, or its complementary polynucleotide.
Said polynucleotide comprise at least 10 contiguous nucleotides of the Nucleotide (expressing through n) of the pleomorphism site (the 101st) that contains in the nucleotide sequence that is selected from nucleotide sequence SEQ ID NOS:1-12.Said polynucleotide are preferably 10-200 Nucleotide, 10-100 Nucleotide more preferably, and 10-50 Nucleotide more preferably.
Each nucleotide sequence is a polymorphic sequence shown in the SEQ ID NOS:1-12.Polymorphic sequence refers to contain the nucleotide sequence of pleomorphism site here, and SNP (SNP) takes place on described pleomorphism site.Pleomorphism site refers to take place on the polymorphic sequence position of SNP.Described nucleotide sequence can be DNAs or RNAs.
In the present invention, each pleomorphism site of polymorphic sequence shown in the SEQ ID NOS:1-12 (the 101st) is relevant with colorectal carcinoma.This passes through to confirm from the dna nucleotide sequence analysis of colorectal carcinoma patient and normal people's blood sample.This analytical results is summarized in the table 1 and 2.
Polymorphic sequence shown in the table 1SEQ ID NOS:1-12 is related with colorectal carcinoma
The characteristic of polymorphic sequence shown in the table 2SEQ ID NOS:1-12
In table 1 and 2, as follows content-defined in the hurdle.
-ASSAY_ID represents the affinity tag title.
-SNP is the polymorphum base of SNP pleomorphism site.Here; A1 and A2 represent low frequency allele (low mass allele) and high frequency allelotrope (high mass allele) respectively; As result, and optionally design for the convenience of testing according to (Sequenom) sequential analysis of homology MassEXTEND (hME) technology.
The representative of-SNP sequence contains the sequence in SNP site, promptly 101 sequences that contain allelotrope A1 or A2.
-on the gene frequency hurdle, cas_A2, con_A2 and Delta represent the absolute value of difference of allelotrope A2 frequency and cas_A2 and con_A2 of allelotrope A2 frequency, the normal group of case group respectively.Here, cas_A2 is (genotype A2A2 frequency * 2+ genotype A1A2 frequency)/(sample number * 2) in the case group, and con_A2 is (genotype A2A2 frequency * 2+ genotype A1A2 frequency)/(sample number * 2) in the normal group.
Every kind of genotypic frequency of-genotype frequency representative.Here; Cas_A1A1, cas_A1A2 and cas_A2A2 are respectively the number that has the individuality of genotype A1A1, A1A2 and A2A2 in the case group, and con_A1A1, con_A1A2 and con_A2A2 are respectively the number of the individuality that has genotype A1A1, A1A2 and A2A2 in the normal group.
The chi-square value that-df=2 representative has two degree of freedom.The chi-value is represented the chi-square value, and the p-value is confirmed based on the chi-value.Chi_exact_p-Value represents the p value of the Fisher rigorous examination of chi square test (chi-square test).When genotypic number less than 5 the time, the possibility of result of chi square test is inaccurate.In this, need to confirm statistical significance (p-value) more accurately through the Fisher rigorous examination.Chi_exact_p-Value is the variable of in the Fisher rigorous examination, using.In the present invention, when p-value≤0.05, thinking genotype and normal group different of case group promptly, has significant difference between case group and the normal group.
-on dangerous allelotrope hurdle,, reference allele (that is, during cas_A2＞con_A2), just regards allelotrope A2 as dangerous allelotrope greater than the allelotrope A2 frequency of normal group when being the allelotrope A2 frequency of A2 and case group.And under opposite situation, then regard allelotrope A1 as dangerous allelotrope.
-dominant ratio (odd ratio) is represented in the case group ratio of dangerous allelic possibility in the dangerous allelic possibility and normal group.In the present invention, adopt Mantel-Haenszel dominant ratio method.CI represents 95% fiducial interval of dominant ratio, adopts (lower limit of fiducial interval, the upper limit of fiducial interval) expression.When 1 drops in the fiducial interval, think the related not remarkable of dangerous allelotrope and disease.
Hardy-Weinberg equilibrated result is satisfied in-HWE representative.Here, con_HWE and cas_HWE represent respectively in normal group and the case group with respect to the Hardy-Weinberg equilibrated degree of deviation.(p-value=0.01 df=1), regarding Hardy-Weinberg imbalance (HWD) as greater than 6.63 value, and is regarded Hardy-Weinberg balance (HWE) as less than 6.63 value based on chi_ value in the chi square test (df=1)=6.63.
The number of the sample that-call rate representative can make an explanation to its genotype is than the total number of samples of using in the experiment.Here, cas_call_rate and con_call_rate represent the ratio of the number of soluble genotypic sample in case group and the normal group and the total number of samples of application (300 individuality) respectively.
-rs represents SNP identification code among the NCBI dbSNP.
Table 1 and 2 has shown the characteristic based on the SNP affinity tag of NCBI build 119 (on February 1st, 2005).
Shown in table 1 and 2, according to the chi square test of the polymorphism mark thing of SEQ ID NOS:1-12 of the present invention, in the 0.0000008-0.049 scope of Chi_exact_p-Value in 95% fiducial interval.This shows in the polymorphism mark thing of SEQ ID NOS:1-12, significant difference is arranged between the expected value of allelotrope occurrence frequency and the observed value.The dominant ratio scope is 1.30-2.06, and this polymorphism mark thing that shows SEQ IDNOS:1-12 is relevant with colorectal carcinoma.
Therefore, polynucleotide of the present invention can be effectively applied to diagnosis, fingerprinting (fingerprinting analysis) and the treatment of colorectal carcinoma.Particularly, polynucleotide of the present invention can be used as the primer or the probe of colorectum cancerous diagnose.In addition, polynucleotide of the present invention can be used as the antisense DNA or the compsn of colorectal carcinoma treatment.
The present invention also provides allele specific polynucleotide or its complement that is used for diagnosis of colorectal cancer; Itself and the multi-nucleotide hybrid that comprises at least 10 contiguous nucleotides, the said polynucleotide of at least 10 contiguous nucleotides that comprise contain the Nucleotide of the pleomorphism site of the nucleotide sequence that is selected from nucleotide sequence SEQ ID NOS:1-12.
Allelotrope-specific polynucleotide refer to specifically the polynucleotide with each allelotrope hybridization.That is, allelotrope-specific polynucleotide have in the polymorphic sequence of SEQ ID NOS:1-12 the Nucleotide of distinguishing pleomorphism site, and specifically with the ability of each such Nucleotide hybridization.Hybridization is carried out under rigorous condition, and for example, salt concn is no more than 1M, and temperature is not less than 25 ℃.For example, (5mM EDTA pH7.4) is fit to for allelotrope-specific probe hybridization with 25-30 ℃ condition 5 * SSPE for 750mM NaCl, 50mM phosphoric acid Na.
In the present invention, allelotrope-specific polynucleotide can be primer.As it is applied here; Term " primer " refers to single stranded oligonucleotide; It for example, is comprising four kinds of different nucleoside triphosphates and polysaccharase as under appropriate condition; As in the damping fluid of DNA or RNA polymerase or reversed transcriptive enzyme and in the suitable temperature, under template guided, carry out DNA synthetic starting point.The appropriate length of said primer can be according to application aims and difference is generally 15-30 Nucleotide.Normally, short primer molecule needs lower temperature to form stable crossbred with template.Primer sequence need be not complementary fully with template, but must be enough complementary to hybridize with template.Preferably, 3 ' of primer-Nucleotide (n) corresponding (align) of each pleomorphism site of terminal and SEQ ID NOS:1-12.Primer and target dna hybridization that contains pleomorphism site and beginning amplified allele (allelic amplification), wherein primer presents the complete homology with target dna.Primer is paired use, its second primer and opposite strand hybridization.Amplified production obtains through using two primer amplifications, and it means has specific allelic form.Primer of the present invention comprises the polynucleotide passage that is applied to ligase chain reaction LCR (LCR).
In the present invention, allelotrope-specific polynucleotide can be probe.As applied here, term " probe " refers to hybridization probe, that is, can sequence-specifically with the complementary strand bonded oligonucleotide of nucleic acid.
Such probe can be PNAG3, as by Nielsen et al. at Science 254, disclosed among the 1497-1500 (1991).Probe of the present invention is allelotrope-specific probe.In this, when in two members' that are being derived from same species the nucleic acid fragment pleomorphism site being arranged, described probe and the dna fragmentation hybridization that is derived from a member, and do not hybridize with the dna fragmentation that is derived from another member.In the case, hybridization conditions should enough rigorously only be hybridized with an allelotrope according to the significant difference of the intensity for hybridization between the allelotrope with permission.Preferably, the middle part of probe, promptly the 7th of the probe of 15 Nucleotide the, or the 8th or the 9th of the probe of 16 Nucleotide, corresponding with each pleomorphism site of the nucleotide sequence of SEQ ID NOS:1-12.Therefore, can cause significant hybridization difference between the allelotrope.Probe of the present invention can be applicable to and is used for surveying allelic diagnostic method.Said diagnostic method comprises the detection method based on nucleic acid hybridization, for example, and the southern trace.Carry out under the situation based on the detection method of nucleic acid hybridization at the Application of DNA chip, probe can be to be fixed on the form of changing on the substrate of DNA chip.
The present invention also provides the microarray that is used for diagnosis of colorectal cancer, and it comprises polynucleotide of the present invention or its complementary polynucleotide.The polynucleotide of microarray can be DNA or RNA.Described microarray is except comprising polynucleotide of the present invention, and is identical with common microarray.
The present invention also provides the colorectal carcinoma that comprises polynucleotide of the present invention diagnostic kit.This colorectal carcinoma diagnostic kit is removed and is comprised outside the polynucleotide of the present invention, can comprise the necessary reagent of polymerization, for example, and dNTPs, various polysaccharase and tinting material.
The present invention also provides the method for colorectal carcinoma in the diagnosis individuality; It comprises: nucleic acid samples from described individual the separation, and is measured the Nucleotide (n) of at least one pleomorphism site (the 101st) in polynucleotide or its complementary polynucleotide of SEQ ID NOS:1-12.When at least one the dangerous allelotrope that shows in the Nucleotide of at least one pleomorphism site (the 101st) of sample nucleic acid and the table 1 and 2 is identical, confirm that this individuality has higher possibility and is diagnosed as the danger that colorectal carcinoma takes place here.
Nucleic acid samples isolating operation from described individuality can be carried out through the DNA separation method of routine.For example, nucleic acid samples can through use polymerase chain reaction (PCR) amplification target nucleic acid then purifying obtain.Remove outside the PCR, can use LCR (Wu and Wallace, Genomics 4; 560 (1989); Landegren et al., Science 241,1077 (1988)), transcription amplification (transcriptionamplification) (Kwoh et al.; Proc.Natl.Acad.Sci.USA 86; 1173 (1989)), self-sustained sequence replication (self-sustained sequence replication) (Guatelli et al., Proc.Natl.Acad.Sci.USA 87,1874 (1990)) or based on the amplification (NASBA) of nucleotide sequence.The two kinds of methods in back are relevant with isothermal reaction based on isothermal transcription, and produce 30 or 100 times RNA single strand and dna double chain as amplified production.
According to embodiment of the present invention; The operation of measuring the Nucleotide (n) of at least one pleomorphism site comprises; Nucleic acid samples is hybridized on the microarray and detects results of hybridization; On described microarray, fixed the polynucleotide or its complementary polynucleotide that are used to diagnose or treat colorectal carcinoma, these polynucleotide comprise at least 10 contiguous nucleotides among the nucleotide sequence SEQ ID NOS:1-12 and comprise pleomorphism site (the 101st 's) Nucleotide.
Microarray with know in relevant technical field through the probe polynucleotide being fixed on the method for preparing microarray on the substrate.Probe polynucleotide the fixing on substrate that the present invention is relevant with colorectal carcinoma can be used ordinary method and easily carried out.Nucleic acid is also known in relevant technical field in the detection of hybridization on the microarray and results of hybridization.For example, the detection of results of hybridization can be through to produce the mark substance of detectable signal, like fluorescent substance (for example; Cy3 and Cy5); The labeling nucleic acid sample hybridizes to the nucleic acid samples of mark on the microarray, and detects the signal that is produced by mark substance and carry out.
Effect of the present invention
Polynucleotide of the present invention can be used for diagnosis, treatment or the fingerprinting of colorectal carcinoma.
Comprise the microarray of polynucleotide of the present invention and the efficient diagnosis that diagnostic kit can be used for colorectal carcinoma.
The method of analysis of the present invention and the Cancer-Related polynucleotide of colorectum can detect the existence or the danger of colorectal carcinoma effectively.
Preferred implementation of the present invention
Hereinafter, will the present invention more specifically be described through embodiment.Yet the following embodiment that provides only is used for explanation, thereby the invention is not restricted to or do not receive their restriction.
Embodiment
Embodiment 1
In this embodiment; The DNA sample extracts from the blood flow by the individuality of the normal group that is diagnosed as patient's group that colorectal carcinoma patient and the 300 Korea S men and womens that treating form and is made up of 300 Korea S men and womens of the symptom that does not have the colorectal carcinoma patient to organize, and estimates the frequency of occurrences of specific SNP.Said SNP be selected from known DB (NCBI dbSNP:http: //www.ncbi.nlm.nih.com) or (Sequenom:http: //www.realsnp.com/).Be used for the Nucleotide of test dna sample SNP with near the primer of the sequence hybridization SNP that selects.
1.DNA the preparation of sample
The DNA sample extracts from colorectal carcinoma patient and normal people's blood flow.(Molecular cloning:A Laboratory Manual, p 392, Sambrook, Fritsch and Maniatis, 2 according to known process for extracting for DNA extraction
NdEdition, Cold Spring Harbor Press, 1989) and the specification sheets of the commercial reagents box made by Centrasystem carry out.In the DNA sample that extracts, an employing has at least 1.6 purity (through A
260/ A
280Nm ratio mensuration) DNA sample.
2. the amplification of target dna
Target dna is the predetermined DNA zone that comprises SNP to be analyzed, and they pass through pcr amplification.PCR carries out through the ordinary method like following condition.At first, target gene group DNAs is diluted to concentration 2.5ng/ml.Then, prepare following PCR mixture.
Water (HPLC level) 2.24 μ l
10 * damping fluid (15mM MgCl
2, 25mM MgCl
2) 0.5 μ l
DNTP Mix (GIBCO) (every kind of 25mM) 0.04 μ l
Taq?pol(HotStar)(5U/μl) 0.02μl
Forwards/reverse primer mixture (every kind 1 μ M) 0.02 μ l
DNA 1.00ul
TV 5.00 μ l
Here, according to upstream and downstream sequences Design forward and the reverse primer of SNP in the given data storehouse.List in these primers table 3 below.
The PCR condition was following: 95 ℃ of incubations 15 minutes; 95 ℃ of sex change 30 seconds,, extended 1 minute at 72 ℃, and repeat 45 times 56 ℃ of annealing 30 seconds; Finally 72 ℃ of incubations 3 minutes and be stored in 4 ℃.As a result, the target dna fragment that obtains increasing, its length is for being no more than 200 Nucleotide.
3. the Nucleotide of SNP in the analysing amplified target dna fragment
The analytical applications of SNP Nucleotide is carried out from the available homology MassEXTEND of Sequenom (hME) technology in the target dna fragment of amplification.The principle of MassEXTEND technology is following.At first, design ends at the primer (also be called and extend primer) of the previous base of SNP in the target dna fragment.Then, with primer and hybridization of target dna fragment and beginning DNA polymerization.At this moment, polymeric solution contains a kind of reagent (for example ddTTP), and this reagent is mixing and the termination immediately afterwards of first equipotential Nucleotide (for example, A allelotrope) complementary Nucleotide polyreaction.So, when first allelotrope (for example, A allelotrope) when being present in the target dna fragment, has only Nucleotide (for example, T Nucleotide) with this first allelic complementation in the product that obtains from primer extension.On the other hand; When second allelotrope (for example; When G allelotrope) being present in the target dna fragment, be added to 3 ' of primer-end with the Nucleotide (for example, C Nucleotide) of this second allelic complementation; Primer is extended till adding and immediate first equipotential Nucleotide complementary Nucleotide (for example, T Nucleotide) then.Pass through mass spectrometric determination from the length of the product of primer extension.Therefore, can identify the allelotrope that is present in the target dna fragment.Experiment condition is exemplified below.
At first, unreacted dNTP is removed from the PCR product.For this reason, add the pure water of 1.53 μ l, the HME damping fluid of 0.17 μ l, (shrimp alkaline phosphatase mixes the enzyme solution with preparation SAP SAP) and in the 1.5ml test tube to the shrimp alkaline phosphotase of 0.30 μ l.With test tube 5, centrifugal 10 seconds of 000rpm.After this, the PCR product is added the SAP solution pipe, sealing, 37 ℃ of incubations 20 minutes then 85 ℃ 5 minutes, be stored in 4 ℃.
Then, the application target dna fragmentation carries out the homology extension as template.The composition of reaction soln that is used for this extension is following.
Water (nano level pure water) 1.728 μ l
HME extends mixture (10 * damping fluid that contains 2.25mM d/ddNTP) 0.200 μ l
Extend primer (every kind 100 μ M) 0.054 μ l
Thermosequenase(32U/μl) 0.018μl
TV 2.00 μ l
Reaction soln mixes with the target dna solution of previous preparation up hill and dale, and it is centrifugal to carry out spin-down.The test tube or the plate that will contain product mixtures are closely sealed, and 94 ℃ of incubations 2 minutes, then are 94 ℃ 5 seconds, 52 ℃ 5 seconds and 72 ℃ of 40 circulations of 5 seconds, are stored in 4 ℃.Resulting homology extension products washs with resin (SpectroCLEAN).List in the extension primer table of using in the extension 3 below.
Primer that table 3 is used to increase and the extension primer that is used for homology extension target dna s
The Nucleotide of the pleomorphism site in the extension products is used mass spectroscopy, and MALDI-TOF (substance assistant laser desorpted ionize-time of flight method (Matrix Assisted Laser Desorption andIonization-Time of Flight)) detects.MALDI-TOF is according to following operate.When analyte was exposed to laser beam, it flew to the detector that is positioned at a relative side with Ionized matrix (3-hydroxy-picolinic acid (3-hydroxypicolinic acid)) under vacuum state.At this moment, the computational analysis thing arrives the time that detector spent.Material with more little quality arrives detector more soon.The Nucleotide of SNP is measured according to the mass discrepancy between these dna fragmentations and the known SNP nucleotide sequence in the target dna fragment.
Show in the superincumbent table 1 of result and 2 that the Nucleotide application MALDI-TOF of target dna s pleomorphism site detects.Each allelotrope can exist with the form of homozygote in the individuality (homozygote) or heterozygote (heterozygote).According to mendel's law (Mendel ' s Law of inheritance) and hardy-Weinberg law (Hardy-Weinberg Law), the allelic genomic constitution (genetic makeup) that constitutes population keeps with the constant frequency.When genomic constitution has statistical significance, can think that it has biological significance.SNP of the present invention occurs in the colorectal carcinoma patient with statistical level of signification, shown in table 1 and 2, therefore can be used for the diagnosis of colorectal carcinoma effectively.
Sequence table
< 110>Samsung Electronics Co., Ltd (Samsung Electronics Co.Ltd.)
< 120>comprise SNP and with the Cancer-Related polynucleotide of colon, the microarray that contains these polynucleotide and diagnosis examination
Agent box and with the method for this polynucleotide diagnosing colon cancer
<130>PN060547
<160>48
<170>KopatentIn?1.71
<210>1
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or C, pleomorphism site
<400>1
tacattactg?tattgtatac?attttgtcta?tttcttcttt?gattttgttt?ttgtttttat 60
aattatttca?ggtgtgggga?aaaattctgt?ccctgatact?ncatcttgtc?cagaactgga 120
agagctcatt?atttctttat?ttgtactgtt?tttatctatt?catccatagt?gttctcaaca 180
gagctatcaa?aagtattatc?a 201
<210>2
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or G, pleomorphism site
<400>2
tctataccac?agtagtgttg?atctcaagag?cttagcatgt?tggcttaaag?acatccaggg 60
atgaggtgtt?ggagtcagat?tgagtttgaa?tcttagctct?ncttgtatta?ctgtgttatc 120
ttggccaagt?atttaacctc?tctgaaatag?gttttctcag?ggctgtgaag?tttggaagat 180
acataaaagc?ccaaagaaaa?a 201
<210>3
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or C, pleomorphism site
<400>3
ccttaaggtg?gacagaaaat?gaattgtttc?cttcttacca?tctgttattg?tttttgcctg 60
aaggcctgac?tgccttccta?ctccctaata?aacaaagggg?nattctattg?accaattcat 120
tttcaaagag?attttaaaag?gcattaagca?atcaattctc?acatagtgtc?caaattgagt 180
ctctcattca?ttctcttgcct 201
<210>4
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=G or C, pleomorphism site
<400>4
tgacctcagg?tgatccaccc?acctcggcat?cccaaagtgc?tgggattaca?ggtgtgagcc 60
accacaccag?gccttgggta?ccatgccttg?aaccatttca?ntgccttttg?gagatggatg 120
agttgccagc?atccttctta?gatccctata?tatttgttta?tttattgaac?aaatacatat 180
taacttatct?ttttgaccaa?a 201
<210>5
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=G or T, pleomorphism site
<400>5
cccaggattg?gaaatgatgg?atgctttcca?ggggccccga?tccatcatca?gatgaatacg 60
cagccccctc?cccaaggaag?ctcctggttc?attgagatgc?ntaattctct?ccttattttc 120
attactgttt?ctcgtttgta?tggattattt?ttcttcagta?atctgggctt?tacatgactg 180
aataagaaaa?tcatttgttc?a 201
<210>6
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or T, pleomorphism site
<400>6
atttcctgcc?tgtgataaat?gtgtcccaat?atttgtcttt?tggttgttgt?tgttgagaat 60
catttctcat?gttgggaaat?gtgaagtcaa?atagtgtgac?nggacttgct?gaatgattga 120
gtcaaccaca?aatggtattg?tcaaccatgg?ctgttgaatt?aatgagaaca?attaaaactc 180
atttttcaga?ggtcaaaaga?t 201
<210>7
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=G or T, pleomorphism site
<400>7
attagctaaa?cagtttaatg?atgatctgcc?aagaaattga?tgtcagcagt?tagaaaacta 60
aagtcctttt?ttatgcagag?acagcacagt?tggtaaaatt?nttatagttg?acaagttgga 120
aagcagtgca?tgtctctgac?aagacttcag?ctctgtggga?agtgtttgga?aagaaatgga 180
gtgatagtgt?ttgttggcat?t 201
<210>8
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or C, pleomorphism site
<400>8
tgctcatgta?ccttatggat?ttgacccacc?tcattctgga?caaancctca?ggaggatctc 60
ttcagggaca?tgatgcagtt?ttgagactgg?tggagattcg?nacggtatga?agcatttggc 120
ttcttggagt?tttaggtttc?taaattttga?gctccaaggg?tatcacacag?tagctctcat 180
ttaagtgagt?cttctcatgt?t 201
<210>9
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=G or T, pleomorphism site
<400>9
aatatgatgt?cttggatttg?cttcaaaata?atacaagaaa?agggtttgac?ttttggtcag 60
tatatagatg?agtcatgact?gaccatgggg?tgacaattgt?ngaagctgag?ttaggtaccc 120
agaggttcgt?tataatattc?tgtctatgtt?aatctgcaat?atgtctgaca?ttttttataa 180
ttaaaacttc?ttttgaaaag?a 201
<210>10
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=G or T, pleomorphism site
<400>10
attaaaaaac?ctgtattttt?ggatgtattt?ttagaaaaac?agatttacag?gaaacaaacc 60
aaacaaaaag?acttgtggta?caagaaaatt?agaaaataca?ntatatttaa?aatggacgtg 120
ttagcttgtc?ccaggtaaac?tcagttcaaa?atatgggata?aaagagattt?tacttttaac 180
ttcgaacagc?tagagaatga?t 201
<210>11
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or G, pleomorphism site
<400>11
aattggtaat?tactttgatt?aaattaattt?aaaacttggc?agtctgtgga?gacatttttg 60
attgttagag?cttggagggg?ccatccgtgg?gaagaggcca?nggatgctgc?tggaaaccta 120
caatgcccag?gacagccctc?aacaaaaaat?gttctggctt?caaatgtcaa?tagctctgag 180
attgagaaac?cctgttatag?t 201
<210>12
<211>201
<212>DNA
< 213>people
<220>
< 221>variation
<222>(101)
< 223>n=A or G, pleomorphism site
<400>12
aagcagaaga?tgaccagtct?gaggcttcag?ggaagaaatc?tgtgaaggga?gtgtctaaga 60
aatatgttcc?tccacgcttg?gttccagtac?attatggtat?naactttggc?tgctgcctcc 120
tcagcatgaa?ctgtttctct?tttctctgtt?cttggataac?cctgcttatt?ttcatcatgt 180
agatgaaaca?gaagctgagc?g 201
<210>13
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>13
acgttggatg?gagctcttcc?agttctggac 30
<210>14
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>14
acgttggatg?tcaggtgtgg?ggaaaaattc 30
<210>15
<211>19
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>15
tccagttctg?gacaagatg 19
<210>16
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>16
acgttggatg?aaagacatcc?agggatgagg 30
<210>17
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>17
acgttggatg?acttggccaa?gataacacag 30
<210>18
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>18
tgagtttgaa?tcttagctct 20
<210>19
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>19
acgttggatg?tggacactat?gtgagaattg 30
<210>20
<211>31
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>20
acgttggatg?ccttcctact?ccctaataaa?c 31
<210>21
<211>24
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>21
tgaaaatgaa?ttggtcaata?gaat 24
<210>22
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>22
acgttggatg?gggatctaag?aaggatgctg 30
<210>23
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>23
acgttggatg?ttgggtacca?tgccttgaac 30
<210>24
<211>17
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>24
tccatctcca?aaaggca 17
<210>25
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>25
acgttggatg?aaggaagctc?ctggttcatt 30
<210>26
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>26
acgttggatg?tccatacaaa?cgagaaacag 30
<210>27
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>27
ctcctggttc?attgagatgc 20
<210>28
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>28
acgttggatg?ggttgactca?atcattcagc 30
<210>29
<211>31
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>29
acgttggatg?ctcatgttgg?gaaatgtgaa?g 31
<210>30
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>30
actcaatcat?tcagcaagtc?c 21
<210>31
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>31
acgttggatg?atgcagagac?agcacagttg 30
<210>32
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>32
acgttggatg?gtcttgtcag?agacatgcac 30
<210>33
<211>22
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>33
agacagcaca?gttggtaaaa?tt 22
<210>34
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>34
acgttggatg?actgtgtgat?acccttggag 30
<210>35
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>35
acgttggatg?tgcagttttg?agactggtgg 30
<210>36
<211>19
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>36
gccaaatgct?tcataccgt 19
<210>37
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>37
acgttggatg?ataacgaacc?tctgggtacc 30
<210>38
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>38
acgttggatg?tgagtcatga?ctgaccatgg 30
<210>39
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>39
tgggtaccta?actcagcttc 20
<210>40
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>40
acgttggatg?tttacctggg?acaagctaac 30
<210>41
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>41
acgttggatg?ccaaacaaaa?agacttgtgg 30
<210>42
<211>24
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>42
gctaacacgt?ccattttaaa?tata 24
<210>43
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>43
acgttggatg?tcctgggcat?tgtaggtttc 30
<210>44
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>44
acgttggatg?gattgttaga?gcttggaggg 30
<210>45
<211>20
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>45
gtaggtttcc?agcagcatcc 20
<210>46
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>46
acgttggatg?agaaatatgt?tcctccacgc 30
<210>47
<211>30
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>47
acgttggatg?aacagttcat?gctgaggagg 30
<210>48
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>primer
<400>48
ggttccagta?cattatggta?t 21
Claims (6)
1. polynucleotide or its complementary polynucleotide of forming by nucleotide sequence SEQ ID NO:1.
2. the polynucleotide of claim 1, it is primer or probe.
3. the microarray that is used for diagnosis of colorectal cancer, it comprises polynucleotide or its complementary polynucleotide of claim 1.
4. the test kit that is used for diagnosis of colorectal cancer, it comprises polynucleotide or its complementary polynucleotide of claim 1.
5. the polynucleotide of claim 1 are used to prepare the purposes of the microarray or the test kit of diagnosis of colorectal cancer.
6. the purposes of claim 5 has been fixed polynucleotide or its complementary polynucleotide of claim 1 on the wherein said microarray.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2004-0011327 | 2004-02-20 | ||
| KR1020040011327 | 2004-02-20 | ||
| KR20040011327 | 2004-02-20 | ||
| KR10-2005-0013395 | 2005-02-18 | ||
| KR1020050013395 | 2005-02-18 | ||
| KR1020050013395A KR101138863B1 (en) | 2004-02-20 | 2005-02-18 | A polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide |
| PCT/KR2005/000465 WO2005079173A2 (en) | 2004-02-20 | 2005-02-19 | A plynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1771337A CN1771337A (en) | 2006-05-10 |
| CN1771337B true CN1771337B (en) | 2012-09-26 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005800001588A Expired - Fee Related CN1771337B (en) | 2004-02-20 | 2005-02-19 | A plynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucle |
Country Status (2)
| Country | Link |
|---|---|
| KR (2) | KR101138863B1 (en) |
| CN (1) | CN1771337B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101148825B1 (en) * | 2005-02-26 | 2012-05-24 | 삼성전자주식회사 | A protein associated with a colon cancer, a polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the same |
| KR101724130B1 (en) * | 2015-06-16 | 2017-04-10 | 연세대학교 산학협력단 | Biomarkers for Diagnosing Intestinal Behcet's Disease and Uses Thereof |
| CN113782114B (en) * | 2021-09-17 | 2024-02-09 | 北京航空航天大学 | Automatic excavating method of oligopeptide medicine lead based on machine learning |
-
2005
- 2005-02-18 KR KR1020050013395A patent/KR101138863B1/en active IP Right Grant
- 2005-02-19 CN CN2005800001588A patent/CN1771337B/en not_active Expired - Fee Related
-
2011
- 2011-09-27 KR KR1020110097575A patent/KR101100437B1/en active IP Right Grant
Non-Patent Citations (3)
| Title |
|---|
| Kawakami K. & Watanabe G..Identification and Functional Analysis of Single NucleotidePolymorphism in the Tandem Repeat Sequence ofThymidylate Synthase Gene.Cancer Res.vol.63.2003,vol.636004-6007. * |
| Reference SNP Id:rs2295706,NCBI Assay Id:ss12673809.NCBI Single Nucleotide Polymorphism.2003,序列. * |
| ReferenceSNPId:rs2295706 NCBI Assay Id:ss12673809.NCBI Single Nucleotide Polymorphism.2003 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1771337A (en) | 2006-05-10 |
| KR101138863B1 (en) | 2013-03-07 |
| KR20110110758A (en) | 2011-10-07 |
| KR101100437B1 (en) | 2011-12-30 |
| KR20060042952A (en) | 2006-05-15 |
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