CN1331755A

CN1331755A - Method for indexing and determining relative concentration of expressed messenger RNAs

Info

Publication number: CN1331755A
Application number: CN99814965A
Authority: CN
Inventors: 卡尔·W·哈塞尔; 布赖恩·S·希尔布什
Original assignee: Digital Gene Technologies Inc
Current assignee: Digital Gene Technologies Inc
Priority date: 1998-11-04
Filing date: 1999-10-14
Publication date: 2002-01-16
Also published as: EP1127159A1; AU1108900A; IL142965A0; EA200100490A1; MXPA01004550A; NO20012203D0; CA2350168A1; JP2002528135A; NO20012203L; WO2000026406A1; KR20010092721A

Abstract

An improved method for the simultaneous sequence-specific identification of mRNAs in a mRNA population allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. In general, the method comprises the formation of cDNA using anchor primers to fix a 3'-endpoint, preparing cRNA, transcribing cDNA from the cRNA and performing two sequence specific PCR amplifications of the cDNA. In preferred embodiments, the method comprises comparing the length and at least part of the nucleotide sequence of the PCR products to expected values determined from a database of nucleotide sequences. The method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions. Also provided are vectors and primers useful for the practice of the improved method.

Description

Retrieval and definite method of expressing the relative concentration of messenger RNA(mRNA) s

The present invention relates to the method for synchronous evaluation of the different mRNAs that express and the measuring method of their relative concentrations.

The complete characteristic of forming organic protein molecule, for the selection that for example improves medicinal design, individual patient optimization treatment, and the research and development of how suitable biological substance be useful.A such characteristic of expressing protein will comprise their identification, the proof of order is determined, their are expressed anatomical site, and they are bioactive illustrates and how these activity are determined the physiological understanding of organism.For the application of medicine, should comprise the information how change of every kind of protein concn makes a response to medicinal or toxic medicament.

Does the scope about this problem that needs us to consider comprise: total how many genes? the problem that has how many genes to be expressed in Mammals does not still solve after 20 years in research at least.The direct research of being devoted to gene expression pattern in different tissues is seldom arranged.Mutational load research (J.O.Bishop, " gene dosage strategy ", cell 2:81-86 (1974); T.Ohta.﹠amp; .M.Kimura, " as the functional organization of the genetic stew of a product of molecular evolution, " nature, 223:118-119 (1971)) shown have 3 * 10 ⁴To 10 ⁵Between indispensable gene.

Before the cDNA clone technology, the information source of genetic expression is in the research of RNA complicacy: it is based on to the similarity measurement on the observation basis in the abundant mixing storehouse with not homospecific RNA molecule (measuring in batch).For expecting outer scope, the complicacy that early stage similar complicacy research is in fact hidden is distorted, Jiu Shi total this true most molecule of forming their mRNA quality in each tissue only accounts for the sub-fraction of its complexity.Afterwards, cDNA clone allowed the application special sequence measuring of individual species (for example, based on) of digital measurement; Therefore, the more modern notion of expressing about mRNA is based on the basis of the actual observation of the RNA species of individuality.

Brain, liver, kidney are measured once by the mammiferous tissue of broad research extremely by similar RNA complicacy.The minimum estimation of complicacy is Hastie and Bishop (N.D.Hastie﹠amp; J.B.Bishop, " three classes are enriched the expression of messenger RNA(mRNA) in the tissue of mouse, " cell 9:761-774 (1976)), they point out 3 * 10 ⁹Have 26 * 10 in the genome of base pair Rodentia ⁶Nucleotide is expressed in brain, and 23 * 10 ⁶In liver, expressed 22 * 10 ⁶In kidney, expressed, simultaneously almost completely overlapping in RNA sets.This has shown a very tissue-specific mRNAs of small number.Yet experiment shows that these numbers have been underestimated obviously, because being proved to be in brain, those many mRNA molecules that might enrich under the detectability of this early stage research express, but can not be detected in liver and kidney.Many other researchist (J.A.Bantle and W.E.Hahn, " complicacy of polyadenylic acid RNA and characteristic in the brain of mouse " cell .8:139-150 (1976); D.M.Chikaraishi, " complicacy of Yeast Nucleic Acid in tenuigenin polyadenylic acid and the no adenylic acid (AMP) mouse brain " biological chemistry 18:3249-3256 (1979)) 100-200 in brain * 10 have been measured ⁶The similar complicacy of Nucleotide, it is than at the low 2-3 of numerical value of liver and kidney estimation doubly.For the mRNAs of brain, 50-65% in liver and kidney is can not be detected.These numerical value obtain the support (R.J.Milner and J.G.Sutsliffe, " expression of gene in the mouse brain " nucleic acids research, 11:5497-5520 (1983)) of numeral clone research.

The similarity measurement of a large amount of mRNA shows that average mRNA length is between 1400-1900 Nucleotide.By the careful blot hybridization of promise (Milner and Sutcliffe, supra) in the system digits analysis with 200 brain cDNAs measure R NA length of selecting at random, find when the mRNA length data is weighed with the advantage of RNA, mean length is 1790 Nucleotide, and this is the same with the determined numerical value of similar measurement.Yet the most of mRNAs that form brain mRNA complexity have the mean length of 5000 Nucleotide.Not only brain RNAs is long more rare more, and it also is inclined to the brain specificity, and the brain mRNAs that has superiority more simultaneously more generally expresses and be short more on mean length.

These are derived from the length of the brain mRNA that sequence has been determined and further confirm (J.G Sutcliffe, the year of " at the mRNA of mammalian central nervous system " neuroscience is looked back 11:157-198 (1988)) by nearest about the notion of mRNA length.Therefore, 1-2 * 10 ⁸Nucleotide complexity and 5000 average mRNA length of Nucleotide and estimation in brain, express 30,000mRNAs adapts, these mRNA that in brain, express about 2/3 in liver and kidney, detect less than.Clearly brain has occupied sizable part of mammalian tissues specific gene.Most of brain mRNAs express with lower concentration.Do not have the measurement of total Mammals mRNA complicacy, 5000 nucleotide pairs are that the estimation of a suitable mRNA length also is ignorant in non-nervous tissue.Rational estimated value for total gene dosage may be between 50,000 to 100,000.

Understand by one on physiologic function chemistry, needing most what advance development is the coded a series of protein sequences of the cell type that each genome is expressed in various cell types that add by genome.Because in genome sequence, there is interference sequence to have (intron), only just be not easy to infer protein sequence at present from gene from cDNA.Even a complete nucleotide sequence of mammalian genes group can not be represented the feature of its expressed sequence.Therefore, need the strategy process collecting transcription sequence and prove the system in their expression sites.Such strategy process sequence difference in determining brain is useful especially in expressing.It is an inevitable final purpose that such research obtains the result, just, removes all mRNAs of recognition and verification.Because various mRNAs have different advantages with a large amount of mRNA by the uniqueness of many unique tissue expressions, are difficult so remove to obtain such the possibility of result.Make people obtain the description of a more reliable gene space three-dimensional of progressive for obtaining such effort that the result did.

The research of in Craig Venter laboratory, carrying out (M.D, Adams etc., " the complementary DNA order-checking: the expressed sequence tag and the Human Genome Project, " science 252:1651-1656 (1991)); M.D.Adamse etc." Sequence Identification of 2,375 human brain genes " natural 355:632-634 (1992)) caused the people brain mRNAs the cDNA that selects at random clone separation, they 3 '-terminal weak point the single passage that is approximately 300 base pairs (single-pass) sequence determine and in these 2500 compile as " label of expressed sequence (EST) " database.Although these databases are useful, can not provide any information of differential expression.Therefore, based on their comprehensive representation pattern in brain region and other tissue, and based on on the various physiology for example or the various examples of the influence of state on the pathology or pharmacological agent react, it is important removing to discern these genes, rather than their expression in a single organization

Other work mainly concentrates on and utilizes polymerase chain reaction (PCR) to set up a database.(J.G.K.William etc., " is useful as genetic marker by the amplification of random primer dna polymorphism " nucleic acids research .18:6531-6535 (1990) and Welsh﹠amp such as Williams; McClelland (J.Welsh and McCelland, " with the genomic imprinting method of Matrix of Pairwise associating of arbitrarily primed PCR and primer " nucleic acids research .18:7213-7218 (1990)) shows the primer of selecting the single 10-aggressiveness of sequence (mer) at random, the shelf primer of any 10-mer for example, when being used in when carrying out PCR the arrangement of a PCR product of generation as the complex DNA template of people, plant, yeast or bacterial genomes DNA.The primer fact is proved to be and relates to not exclusively complementation between primer and template DNA like this.Can suppose that part mispairing primer binding site is a stochastic distribution in genome.Once in a while, these two sites are in opposite direction, and mutual distance is enough near, to such an extent as to can produce PCR product band.Average total 8-10 product, its size changes between approximately O.4-4kb, and each primer is had a different mobility.The PCR product is arranged in the individuality of same species and has shown difference.These authors propose the information that single arbitrary primer can product restriction fragment length polymorphism (RFCP) sample is used for genome research.Other people has also used this technology (S.R.Woodward etc., " the stochastic sequence oligonucleotide primer detects isolated polymorphic dna product in inbred mouse is ", Mammals heredity 3:73-78 (1992); J.H.Nadeau etc., " to the multidigit point mark of musculus cdna group analysis: based on the pcr amplification on single primer basis of any nucleotide sequence ", Mammals heredity 3:55-64 (1992)).

Two study group (J.Welsh etc., " the PCR trace of the arbitrary primer of RNA ", nucleic acids research, 20:4965-4970 (1992); P.Liang and A.B.Pardee, " showing Eukaryotic messenger RNA(mRNA) ", science 257:967-971 (1992) by polymerase chain reaction method difference) revised the sort of method to compare the mRNA storehouse.In the research of Liang and Pardee, this method is known as mRNA difference and shows that it is used to by two relevant cell types, and normal and oncogene mouse A31 cell is with the mRNAs storehouse of relatively expressing.For each experiment, they with any 10-mer as the tail complementary oligonucleotide of 5 ' one primer and one and an inferior cover poly A (poly A) as 3 '-anchor primer, from the cDNA of two cell type preparations, and carry out pcr amplification under the condition of 35S-dNTPs existence.Product is analyzed on sequencing gel, and scope is observed at 50-100 band of 100-500 Nucleotide.These bands infer and derive from the cDNAs amplification, they are with to contain 3 ' of 3 ' one anchor primer complementary mRNAs-end corresponding with part mispairing 5 '-primer sites, and this is as being observed in the genomic dna template.Right for each primer, be similar from the pattern of the band of two cDNAs amplification, it is undistinguishable that about 80% distribution density is arranged in the band.On the template of the PCR sample of or other, it is more intensive that some bands are arranged; Two samples only have in one, and it is detectable that several bands are seldom arranged.

Further study (P.Liang etc., " by distribution and the clone of difference display method eukaryotic mrna s: purify and optimize; " nucleic acids research .21:3269-3275 (1993)) prover to lower concentration input RNA work (although it is not quantitative to more rare species), and specific residue mainly is present in the last Nucleotide of 3 ' one anchor primer, at least the difference that is identified identification detects 1/3 corresponding with differential expression RNAs in the PCR product, and at least 25% false positive rate is arranged simultaneously.

If all mammiferous 50,000-100,000mRNAs is applicable to the method for this arbitrarily primed PCR, need so about 80-95 5 ' arbitrary primer and 12 3 ' anchor primers in about 1000 PCR groups and gel to draw a possibility, calculate by Poisson's distribution, have about 2/3 will be identified among these mRNAs.

Because following reason, it is impossible that all mRNAs are fit to adopt this method to detect.In a such detection, require a mRNA to be presented on the surface, it must have enough advantages to produce signal in radioautograph, and include one in its 3 '-sequence of 500 Nucleotide that end works as the combination of a mispairing primer and design of primers site.An independent mRNAs takes advantage more, and it might produce a product more.Therefore, may produce band with a large amount of different arbitrary primer advantage species.Because the attribute based on the latter on the basis of arbitrary primer selection contains a uncertain chance key element, it is difficult obtaining the possibility of result by the arbitrary primer method.And because information can pass to another laboratory from a laboratory, reliably the mispairing primer must have high performance reproducibility under different experiment conditions and condition with different PCR instrument, and the while, the result had change a little under reaction conditions.Basis as the mispairing design of primers is seldom understood, and this is to pass through Liang﹠amp; The Pardee difference display method obtains the drawback that data are set up a database.

U.S. Patent number 5,459,037 (' 037) and 5,807,680 (' 680) described the method that an improved mRNA species difference shows, this method has reduced by the 5 '-terminal uncertain aspect that produces, and allow data in the different groups fully repeatably.This method is not fixed against the unrepeatable mispairing primer of potential, has reduced and has carried out the required PCR group of end-to-end and the quantity of gel, and made double-stranded sequence data promptly be accumulated.And this improved method has also reduced the quantity of the signal that occurs when same species mRNA obtains.He ' 680, ' 037 patent in this literary composition by incorporated by reference and as a disclosed part.

Still there are further improved needs in disclosed method in the patent of ' 037.For example, by reducing in complementary DNA molecule synthesis and PCR the wrong design of primers between the reaction period, the specificity of this method might be enhanced.And this technology can be further optimized, so that it has more repeatability, more responsive, easier application.More superior part is, this technology will provide the sequence of utilization from forming database and obtaining, browse the few nucleotide of GenBank for example comes recognition sequence identity and similarity according to base, with for example BLASTN and the such computer software of BLASTX ability.

We have developed out improving one's methods that a kind of synchronous sequence-specific identification of carrying out mRNAs in a mRNA storehouse identifies.This improved method is by 1) sequence of the part Nucleotide that a+b is long, wherein a is the length in the base of restriction enzyme enzyme recognition site, b is the number of degeneracy base, 6 〉=b 〉=3 here, with 2) partial sequence is apart from the distance of poly (A) tail, on the identity of determining and the basis of address mRNAs classified.Representative identity and address are determined that by a partial sequence this partial sequence comprises one four base recognition site and four degeneracy bases of restriction enzyme.In a preferred embodiment, the recognition site of this restriction enzyme is MspI, and partial sequence is C-C-G-G-N ₁-N ₂-N ₃-N ₄Because this method depends on the nucleotide sequence of a mRNA, rather than its advantage in a specific tissue, so this method can be used to explain all mRNAs in the above concentration appearance of detection threshold.Compare with the RAP-PCR method with the difference demonstration, do not have uncertain aspect for 5 '-terminal generation.

A preferred embodiment (Fig. 1) of the method according to this invention contains in the cDNA library that produces from each mRNA sample, nearly all poly (A) in initial RNA sample ⁺3 '-farthest terminal copy that begins to poly (A) tail from the MspI site of farthest side of mRNAs, the relative concentration of itself and initial mRNAs almost is corresponding.Because two ends of each species insertion sequence all by the sequence of mRNAs self accurate limit, so for each species, segmental length is consistent, this makes them that a concrete visible band be arranged on gel.No matter mRNA's is how tissue-derived, these length all are constant, and this is an important key concept of this method.The messenger RNA(mRNA) s that lacks the MspI recognition sequence is not described, and they are rare comparatively speaking.These mRNAs are hunted down by the method for using the different restriction enzymes that can discern difference four base recognition sequences.

Another aspect of an embodiment so of the present invention is the sequence that application and 3 '-restriction endonuclease sites close on, and in a preferred embodiment, classify to cDNAs at least two successive PCR steps in a MspI site.First PCR step is to utilize a primer and derive from for example carrier pBC SK ⁺Sequence annealing, but the CGG by the MspI site of not regenerating remove to comprise insertion sequence first in abutting connection with Nucleotide (N ₁) extend.The mRNAs storehouse of this step with beginning is separated into 4 inferior storehouses.In second PCR step, each in 4 inferior storehouses that produce by first PCR step further is divided into 64 parts, by with the insertion primer (N that more can invade ₁N ₂N ₃N ₄) be divided into 256 Ya Yaku.In last PCR step,,, make a fluorescence labels be incorporated into product and detect by the laser apparatus induced fluorescence by with fluorescent mark 3 ' PCR primer.

In a preferred embodiment, but for example the such isolation technique of the electrophoresis PCR product molecule that is used to evaluation of markers become measured intensity and and the corresponding band that can distinguish of measurable length arranged.Suitable isolation technique comprises gel electrophoresis, capillary electrophoresis, the suitable isolation technique of other that HPLC, MALDI mass spectroscopy and this field are in common knowledge, these technology have single base resolving power in 50-500 base scope ability is that the present invention is included.

Therefore in a preferred embodiment, each last PCR reaction product is a designated identity and address, and this is based on and contains four base restriction endonuclease sites and four brief bases (C-C-G-G-N for example ₁-N ₂-N ₃-N ₄) a 8-nucleotide sequence and terminal first A with at the polyA tail of the 3 ' end of mRNA the courier between begin from the junction on the basis of distance of that sequence.When experiment is determined, or when being known by the segmental nucleotide sequence of PCR product that a database sequence is determined, this fragment is by with reference to as a digital sequence label (DST): that is to say, derive by the inventive method and come one 3 '-terminal EST (expressed sequence tag).Detect the intensity of the separated segmental band of mark PCR product with a suitable method, preferably the fluorescence of induced with laser (but radioactivity or magnetic mark and detection may be used to) is by quantitatively, and with being that the specified address of PCR product fragment stores each PCR product fragment in a database.The separated segmental intensity of mark PCR product and proportional with the amount of the corresponding initial mRNA of PCR product fragment.

Generally speaking, method of the present invention comprises:

(a) prepare a double-stranded cDNA storehouse with a blended anchor primer from a mRNA storehouse, each anchor primer contains one 5 ' terminal and one 3 ' end, and comprises one section sequence of (i) 7-40 T residue; (ii) discern the site of being cut by first restriction enzyme of 6 above bases, this site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, first stuffer are positioned toward by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer insert be used to discern 6 above bases by between first restriction enzyme cleavage site and the T residue sequence; (v) from by-V,-V-N, the phase transformation residue of each anchor primer of selecting in the group that forms with-V-N-N is positioned in 3 '-end, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G, N is from by A, C, the deoxyribonucleotide that screens in the group that G and T form, this mixture comprises the possible anchor primer that contains all V and N.

(b) downcut double-stranded cDNA storehouse with first restriction enzyme and second restriction enzyme, 4-nucleotide sequence of this second restriction enzyme identification has formed a double-stranded cDNA library of molecules that first and second ends are arranged respectively.

(c) derive from step (b), each double-stranded cDNA molecule is inserted into into carrier, on direction, be anti-meaning for the special promotor of phage, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ' and the 3 ' flanking vector that inserts the significant chain 5 ' of cDNA-end and sense strand 3 '-end, and the said structure that one 3 ' flanking sequence arranged has 15 length of nucleotides in said first restriction endonuclease sites and qualification at least between transcription initiation site in described promotor.

(d) insert the carrier transformed host cell with the cDNA that is cut and produce the carrier that contains clone's insertion.

(e) with at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the special promoter sequence of phage, but the structure storehouse that the digestion with restriction enzyme of sequence produces in step (c) on the energy identification carrier, so just produced the linear segment of the cDNA molecule that contains insertion, the linear fragment of Chan Shenging has 15 Nucleotide holding second end of double-stranded cDNA molecule at least at carrier 5 ' like this.

(f) by incubation linear fragment and the special RNA polymerase of phage that has initial from the special promoter transcription ability of phage, produced the preparation of the cRNA that antisense cRNA transcribes.

(g) long with a ThermoScript II and a 15-30 Nucleotide, and, produce the first chain cDNA by transcribing cRNA by 5 ' the reverse transcription primer of forming with 5 ' flank body sequence complementary nucleotide sequence (RT).

(h) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA.The polymerase chain reaction is that 15-30 Nucleotide is long with first 3 ' PCR primer for the first time, it and determine 3 ' flanking vector sequence complementation between transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, first 5 ' PCR primer has been defined-N ₁3 ' the end of forming, wherein " N " is among four kinds of deoxyribonucleotide A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier sequence complementation of 5 ' flank, simultaneously, the complementation of first 5 ' PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, is used in four inferior storehouses of difference each at different primers of this first 5 ' PCR primer.

(i) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product.Second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, it and determine 3 ' flanking vector sequence complementation between transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in the step (h), x is from 1 to 5 integer, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, simultaneously complementary the extension with Nucleotide quantity of primer is equivalent to the special insertion Nucleotide that cRNA is inserted in " x "+1, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and has 4 for each inferior storehouse in the inferior storehouse of first cover in second series ^xIndividual inferior storehouse.

(j) analyze of the displaying of the second cover PCR product with the distinguished sequence of 3 ' of the mRNAs that produces a representative and in the mRNA storehouse, occur-terminal.

In a preferred embodiment, a biotin moiety is joined on the anchor primer by conjugation, preferably joins 5 ' of anchor primer-end to.In such an embodiment, contact by the cDNA of first qualification and the substrate of a streptavidin bag quilt, first limits cDNA and is separated in the residuum of the cDNA from step (b).The substrate of suitable streptavidin bag quilt comprises microtiter plate, PCR pipe, polystyrene spheres, paramagnetic polymeric beads and paramagnetic sintered glass particulate.The substrate of the streptavidin bag quilt of an excellent usefulness be the paramagnetic polymeric beads suspension (Dynal, Inc., Lake Success, NY).

In one embodiment, first 5 ' PCR primer connects by resistance phosphodiesterase bonding at 3 ' 3 terminal Nucleotide, and preferably the thiophosphatephosphorothioate bonding connects.In an other embodiment, second 5 ' PCR primer connects by resistance phosphodiesterase bonding at 3 ' 3 terminal Nucleotide, and preferably 3 Nucleotide of 3 ' end of first and second two 5 ' PCR primers are connected by thiophosphatephosphorothioate.

Typically, in second PCR reaction primer is engaged a last fluorescence labels by conjugation.A suitable fluorescence labels is to select from the group that is made up of following material,

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene (xanthen)-3-one, the 6-carboxylic acid, 3 ', 6 '-dihydroxyl-6-Fluoresceincarboxylic acid (6-FAM, ABI);

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 5-carboxylic acid, 3 ', 6 '-dihydroxyl-5-Fluoresceincarboxylic acid (5-FAM, molecular probe);

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 3 ', 6 '-dihydroxyl fluorescein (FAM, molecular probe);

9-(2,5-dicarboxyl phenyl)-3,6-two (dimethylamino)-xanthene (xanthylium) (6-carboxyl tetramethylrhodamin (6-TAMRA), molecular probe);

(2-carboxyl phenyl)-(rhodamine is green for xanthene for 3,6 one diamino-9- ^TM, molecular probe);

Spiral [isobenzofuran-1 (3H), 9 '-xanthene]-6-carboxylic acid, 5 '-two chloro-3 ', 6 '-dihydroxyl-2 ', 7 '-dimethoxy-3-oxo-(JOE, molecular probe);

1H, 5H, 11H, 15H-xanthene be (xantheno) [2,3,4-ij:5,6,7-i ' j '] two quinolizines-8-also ,-(2,4-two sulfur phenenyls)-2,3,6,7,12,13,16,17-octahydro one, inner salt (TexasRed, molecular probe);

6-((4-4-two fluoro-5,7-dimethyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino) caproic acid (BODIPY FL-X, molecular probe);

6-((4,4-two fluoro-1,3-dimethyl-5-(4-aminomethyl phenyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino) caproic acid (BODIPY TMR-X, molecular probe);

6-(((4-(4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) phenoxy group) ethanoyl) amino)-caproic acid (BODIPY TR-X, molecular probe);

4,4-two fluoro-4-boron-3a, 4a-phenodiazine-s-indacene-3-valeric acid (BODIPY FL-C ₅, molecular probe);

4,4-two fluoro-5,7-dimethyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid (BODIPY FL, molecular probe);

4,4-two fluoro-5-phenyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid (BODIPY581/591, molecular probe);

4,4-two fluoro-5 (4-phenyl-1,3-butadiene base)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid (BODIPY 564/570, molecular probe);

4,4-two fluoro-5-styryl (styryl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid;

6-(((4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) styryl oxygen (styryloxy)) ethanoyl) hexosamine (BODIPY 630/650, molecular probe);

6-(((4,4-two fluoro-5-(2-pyrryl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) styryl oxygen) ethanoyl) hexosamine (BODIPY 650/665, molecular probe);

9-(2,4 (or 2,5)-dicarboxyl phenyl)-3,6-two (dimethylamino)-xanthene, inner salt (TAMRA, molecular probe).

Other fluorescent mark applicatory comprises 4,7,2 ', 4 ', and (" HEX ", ABI), " NED " (ABI) with 4,7, (" TET " is that this field is known ABI) to 2 ', 7 ' tetrachloro-6-Fluoresceincarboxylic acid to 5 ', 7 ' chlordene-6-Fluoresceincarboxylic acid.

Representative is, the phase transformation residue in step (a) has the 3 ' end of one-V-N-N, in another embodiment, the phase transformation residue in step (a) have one-V or-3 ' end of V-N.

In a preferred embodiment, " x " is 3 in step (i), and preferable is that the phase transformation residue in step (a) is-V-N-N that " x " is 3 in step (i).

Representative is that each anchor primer has 8-18 T residue in the T residue sequence.In a preferred embodiment, each anchor primer has 18 T residues in the T residue sequence.In other embodiments, in the T residue sequence, each anchor primer has 8-18 T residue, preferred 8-16 T residue, more preferably 8-14 T residue, most preferably 8-12 T residue.In a further advantageous embodiment, in the T residue sequence, each anchor primer has 12 T residues.

Representative is that first anchor primer stuffer is that 14 residues are long.In one embodiment, first stuffer has A-A-C-T-G-G-A-A-G-A-A-T-T-C (SEQ IDNO:1) nucleotide sequence.In another preferred embodiment, first stuffer has G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO:2) nucleotide sequence.

Representative is that the special promotor of phage is screened from being started the molecular group by T3 promotor, T7 promotor and SP6.Preferably, the special promotor of phage is the T3 promotor.

In one embodiment, the primer sequence of transcribing design from the cDNA of cRNA is A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO:14).In another embodiment, the primer sequence of transcribing design from the cDNA of cRNA is A-G-C-T-C-T-G-T-G-G-T-G-A-G-G-A-T-C (SEQ ID NO:28).In another embodiment, the primer sequence of transcribing design from the cDNA of cRNA is T-C-G-A-C-T-G-T-G-G-T-G-A-G-C-A-T-G (SEQ ID NO:35).

In one embodiment, carrier is the plasmid pBC SK+ with ClaI and NotI cutting, in step (h) and 3 ' PCR primer (i) be G-A-G-C-T-C-C-A-C-C-G-C-G-T (SEQID NO:47).In another embodiment, carrier is the plasmid pBCSK+ with ClaI and NotI cutting, in step (h) and 3 ' PCR primer (i) be G-A-G-C-T-C-G-T-T-T-T-C-C-C-C-A-G (SEQ ID NO:48).

Representative is, first restriction enzyme of 6 above bases of identification is from by AscI, BaeI, FseI, NotI, PacI, PmeI, PpuMI, RsrII, SapI, SexAI, SfiI, SgfI, SgrAI, SrfI screens in the group that Sse8387I and SwaI formed.Preferred first restriction enzyme of 6 above bases of identification is NotI.

Representative is, second restriction enzyme of a 4-nucleotide sequence of identification is from by MboI, DpnII, Sau3AI, Tsp509I, HpaII, BfaI, Csp6I, MseI, HhaI, NlaIII, TaqI, MspI screens in the group that MaeII and HinP1I formed.Preferred second restriction enzyme of a 4-nucleotide sequence of identification is MspI, Sau3AI and NlaIII.

Representative is that the restriction enzyme that is used in the step (e) has an identification that contains a nucleotide sequence of the 4-nucleotide sequence that is used in second restriction enzyme in the step (b).In one embodiment, this second restriction enzyme is MspI, and the restriction enzyme that is used in the step (e) is SmalI.In another embodiment, this second restriction enzyme is TaqI, and the restriction enzyme that is used in the step (e) is XhoI.In another embodiment, this second restriction enzyme is MaeII, and the restriction enzyme that is used in the step (e) is AatII.

Representative is, the carrier of step (c) is a circular DNA molecule form, it has the first and second carrier restriction endonuclease sites at carrier padding sequence flank, and it further is included in the step of the first and second carrier restriction endonuclease sites with the digested vector of restriction endonuclease cut vector.Preferably, the padding sequence of this carrier includes an interior carrier filling restriction endonuclease sites between the first and second carrier restriction endonuclease sites.

A proper host cell is intestinal bacteria.

Representative is that step (e) comprises the vector digestion of filling the restriction endonuclease sites cut vector with a restriction enzyme in interior carrier.

Representative is that the restriction enzyme that is used in the step (e) also cuts the carrier of filling restriction endonuclease sites in interior carrier.

For other restriction enzyme, under the situation of the suitable restriction enzyme that does not possess a hexabasic basic recognition site that contains inside four base recognition sites, total strategy that pSK carrier of linearizing adopts comprises the steps: that (i) separately contains the segmental plasmid of insertion and become two portions, first part uses restriction enzyme SalI to cut with restriction enzyme XhoI cutting, second section; (ii) after cutting the reorganization first and second parts; (iii) separately the part of reorganization becomes three parts, and with first part in three parts of the restriction enzyme HindIII cuttings, with in three parts of the restriction enzyme BamHI cuttings second part, the 3rd part is cut with restriction enzyme EcoRI; (iv) digestion back reorganization is these three parts, so that produce a segmental storehouse of linearizing, nearly total 1/6 is corresponding with may make up the product that enzyme cuts with each in this storehouse.

Representative is to have been enriched for the mRNA storehouse of poly adenosine mRNA species.

Representative is that the compartment analysis of amplified fragments is undertaken by the electrophoresis showed product in step (j).Preferably, be ratio in the intensity of the product that shows behind the electrophoresis abundance of the corresponding mRNAs of product almost and in original mixture.In a preferred embodiment, after present method further was included in electrophoresis, the intensity that corresponds to mRNA from product was determined a step of each mRNA relative abundance original mixture.

Representative is with the segmental step of electrophoresis compartment analysis polymerase chain reaction (PCR) amplification product, to be included in the segmental electrophoresis on a plurality of gels.

In one embodiment of the invention, present method further comprises the steps:

(k) wash-out at least one with derive from the corresponding cDNA of mRNA in the electrophoretogram, the band of 3 ' of the representative mRNAs that wherein occurs in sample-end is shown;

(l) the isolating PCR product of amplification in a polymerase chain reaction;

(m) the isolating PCR product of clonal expansion enters in the plasmid;

(n) production derives from the PCR product corresponding DNA of plasmid with clone and separate;

(o) the PCR product of order-checking clone and separate.

An alternative embodiment of the invention comprises following steps:

(a) separate a mRNA storehouse;

(b) with a blended anchor primer, from double-stranded cDNA storehouse of mRNA storehouse preparation, each anchor primer has one 5 '-terminal and one 3 '-end and comprises: (i) from the residue sequence of 7-40T; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer relevant 5 '-end that is positioned toward the site of being cut by first restriction enzyme; (iv) second stuffer inserts 6 above bases of identification by between the sequence of first restriction enzyme inscribe cleavage site and T residue; (V) phase transformation residue-V-N-N is positioned in 3 ' of each anchor primer-end, wherein V is the deoxyribonucleotide that screens from the group that is made up of A, C and G, N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N.

(c) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of second restriction enzyme identification has formed a double-stranded cDNA library of molecules that first and second ends are arranged respectively.

(d) derive from each double-stranded cDNA molecule of step (b) and inserted into carrier from a direction, for a T3 promotor is significant, it has formed in carrier and has contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', the 3 ' flanking vector that inserts cDNA sense strand 5 '-terminal and sense strand 3 '-end, and the said structure that one 5 ' flanking sequence arranged has 15 length of nucleotides in said first restriction endonuclease sites and qualification at least between transcription initiation site in described promotor.

(e) insert the carrier transformed into escherichia coli with the cDNA that is cut and produce the carrier that contains clone's insertion.

(f) with not discerning or, so just produced the linear segment of the cDNA molecule that contains insertion in step (c) in the structure storehouse that at least a digestion with restriction enzyme of cDNA molecule that inserts or the sequence in the T3 promotor produces.

(g), produced the preparation of the cRNA that significant cRNA transcribes by incubation linear fragment and a T3RNA polysaccharase that has initial from T3 promoter transcription ability.

(h) long with a ThermoScript II and a 15-30 Nucleotide, and, produce the first chain cDNA by transcribing cRNA by 3 ' the reverse transcription primer of forming with 3 ' flank body sequence complementary nucleotide sequence (RT).

(i) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA.The polymerase chain reaction is that 15-30 Nucleotide is long with first 3 '-PCR primer for the first time, and it is near first restricted interior 3 ' flanking vector sequence complementation of cutting enzyme site 3 ', and first 5 ' PCR primer has been defined-N ₁3 ' the end of forming, wherein " N " is among four kinds of thymus nucleic acid A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier preface complementation of 5 ' flank, simultaneously, the complementation of first 5 '-PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, in different primers of this first 5 ' PCR primer are used in four inferior storehouses of difference each.

(j) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product.Second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, and it is near first 3 ' flanking vector sequence complementation of cutting enzyme site 3 ' in restricted, and second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (i), x is from by selecting 3 and 4 groups that form, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, primer complementation simultaneously extends into the special insertion Nucleotide of Nucleotide quantity for the cRNA of " x "=1, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and has 4 in the inferior storehouse of second series ^xYa Ku.

(k) analyze the second cover PCR product has produced the distinguished sequence of 3 ' of mRNAs that a representative occurs-terminal in the mRNA storehouse displaying.

Typically, the mixture of 48 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:5).In a preferred embodiment, the mixture of 48 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:8).

Representative is that the mixture of 12 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO:4).In a preferred embodiment, the mixture of 12 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID No:7).

Representative is that the mixture of 3 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ IDNO:3).In a preferred embodiment, the mixture of 3 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:6).

In a preferred embodiment, first restriction enzyme is MspI, and second restriction enzyme is NotI.

Representative is that first 5 ' PCR-primer is G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:22).

In a preferred embodiment, 3 ' PCR primer is that the nucleotide sequence conjugation of SEQ ID NO:47 connects a fluorescence labels in second polymerase chain reaction, and more preferably the nucleotide sequence of SEQ IDNO:47 links to each other with the 6-FAM conjugation.

Suitable value is the integer from 1-5 in step (i), and more preferably, " x " is 3 in step (i).

Representative is that the method for the contact that changes on the variation of detection mRNA expression pattern in a tissue and physiology or the pathology comprises following a few step:

(a) obtain normal or neoplastic first sample that does not have physiology or pathological change;

(b) from first sample, separate a mRNA storehouse;

(c) determine the pattern that mRNA expresses in first sample of tissue by (a)-(j) step of carrying out in total method, produce the demonstration of first distinguished sequence product of 3 '-end of representing the mRNAs that in first sample, occurs;

(d) acquisition has second sample of physiology or pathology alternative construction;

(e) from second sample, separate a mRNA storehouse;

(f) determine the pattern that mRNA expresses in second sample of tissue by (a)-(j) step of carrying out in total method, produce the demonstration of first distinguished sequence product of 3 '-end of representing the mRNAs that in second sample, occurs;

(g) more a plurality of demonstrations change the influence to mRNA expression pattern in tissue to determine physiology or pathology;

More representative plural sample, preferred concrete sample is 3, and preferred is 4 samples at least, and sample repeatedly is extracted and compares.

Representative is, the variation on physiology or the pathology be from by senile dementia, Parkinson's neurological dysfunction, local asphyxia, be addicted to drink, be addicted to drug, screen the group that schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, dysthymia disorders, bipolar manic depressive disorder are formed.

Representative is, variation on physiology or the pathology and study or memory, mood, glutamate dehydrogenase neurotoxic, the dispensing of feed behavior, sense of smell, vision, moving obstacle, virus infection, electroshock, medicine or the toxic side effect of medicine are relevant.

Representative is that the variation on physiology or the pathology is to select from changed, wear out, reached long-term the reinforcement the group that is formed by circadian rhythm.Generally speaking, the variation on physiology or the pathology is from by screening the process that second messenger, hormone, neurotransmitter, somatomedin and neuromodulator are mediated in transcription factor, the cell.Alternative is that physiological or pathological variation is from screening by the basic process that contacts, reaches contact mediation between cytolemma and cytoskeleton outside cell-cells contacting, cell-substrate contact, the cell-born of the same parents.

Preferably, normal or the neoplastic tissue of being made up of cell is from neural system, endocrine system, body (being comprised skin, hair and nail) by cardiovascular systems, lymphsystem, respiratory system, Digestive tract, peripheral nervous system, central nervous system, intestines, and the organ or the organ-tissue of the screening in the group that Skeletal system (comprising bone and muscle), urinary system and reproductive system are formed extract or deutero-.

In a preferred embodiment, the normal or neoplastic tissue of being made up of cell is from by epithelium, endothelium, mucous membrane, body of gland, blood, lymph, conjunctive tissue, cartilage, bone, unstriated muscle, skeletal muscle, cardiac muscle, neurone, neurogliocyte, spleen, thymus gland, hypophysis, Tiroidina, parathyroid gland, adrenal cortex, adrenal medulla, pineal gland, skin, hair, nail, tooth, liver, pancreas, lung, kidney, bladder, ureter, mammary gland, ovary, the uterus, vagina, testis, prostate gland, penis, organ that screens in the group of an E ﹠ E composition or organ-tissue extract or deutero-.

Representative, normal or neoplastic tissue be a structure in the central nervous system of from the group that forms by retina, cerebellar cortex, sense of smell bubble, thalamus, hypothalamus, preceding hypophysis, back hypophysis, hippocampus, nucleus accumbens septi, tonsilla, striatum, cerebellum, brain stem, suprachiasmatic nucleus and backbone rope, screening derive and.

Representative, detect the medicine of a screening and the diverse ways of a known compound effect and form by following steps:

(a) from obtain first duplicate samples of tissue with the organism of the compound treatment of known physiologic function;

(b) from first duplicate samples, separate a mRNA storehouse;

(c) pattern that mRNA expresses in first sample tissue is determined in the demonstration that produces first sequence-specific product of 3 '-end of representing the mRNAs that occurs in first duplicate samples by (a)-(j) step of carrying out in total method.

(d), determine the different of the medicine of screening and a known compound effect from obtaining second duplicate samples the organism of screened drug treating;

(e) from first duplicate samples, separate a mRNA storehouse;

(f) pattern that mRNA expresses in second sample tissue is determined in the demonstration that produces second sequence-specific product of 3 '-end of representing the mRNAs that occurs in second duplicate samples by (a)-(j) step of carrying out in total method;

(g) relatively first and second 's demonstration, so that under the situation that the mRNA species occur, the expression of mRNA is not subjected to the influence of known compound, but is subjected to the influence of screened medicine, therefore, can show the medicine of screening and the difference of known compound effect.

Representative is that screened medicine is to screen from the group that is made up of thymoleptic, Antipsychotic drug, tranquilizer, anticonvulsive drug, oxidase inhibitor, stimulant, anti-Parkinson agent, skeletal muscle relaxant, anodyne, local anesthetic, cholinergic agent, antiviral agent, spasmolytic, steroid and non-steroid anti-inflammatory drug.

More common is, technical term " screened medicine " and " detected medicine " are meant a broad range of useful chemistry and therapeutical agent at this, it comprises the steroid of physiologically active, microbiotic, anti-mycotic agent, antibacterial agent, antitumor agent, anodyne and analgesic compounds, anoretics, anthelmintic, anti-arthritic, the antiasthia agent, anticonvulsive drug, thymoleptic, antidiabetic drug, diarrhea, antihistaminic, antiinflammatory agents, the anti-migraine preparation, anti-many moving sick preparations (animotion sickness preparation), antinanseant, the anti-Parkinson medicine, antipruritic, Antipsychotic drug, febrifuge, antispasmodic, comprise stomach with uropoiesis; Anticholinergic, sympathomimetic, xanthine derivative, cardiovascular preparation comprise calcium channel blocker ,-beta blocker, anti-arrhythmic, hypertension hydragog(ue), blood vessel defeated open cartridge bag draw together whole body, coronal tip and brain; The speed medicament, spices, insect repellent medicine, hair dye or the like of the peptide of slow medicine, parasympatholytic, anti-sympathomimetic, incitantia, tranquilizer, tranquilizer, anaphylactogen, antihistaminic, anti-inflammation medicine, physiologically active and albumen egg, ultraviolet screening of central nervous system stimulant, cough and flu agent, Decongestant, hormone, soporific, immunosuppressor, muscle is like that.Describing those reagent that can consider uses professional technique " physiologically active " to understand from broad sense here, not only refer to Su Sheng is had the medicament of a direct pharmacotoxicological effect, also refer to those field of medicaments useful to indirectly or can be observed the medicament of effect, for example, painted or light tight tissue reaches from organizing radioactive screening of medium ultraviolet or the like for diagnostic purpose.

For example, typical antifungal and mycocide comprise Thiabendazole, chloroxine, amphotericin, candicidin, fungimycin, nystatin, Clodantoin, clotrimazole, ethonam nitrate, miconazole nitrate, pyrrolnitrin, Whitfield's ointment, fezatione, the different thiophene ketone of fluorobenzene, how to hold in the palm ester, three ester spits of fland, zinc and PTO and Sodium Pytithione.

Steroid comprises cortisone, the deoxidation cortisone, the fluorine acetonide, fluohydrocortisone, the oxalic acid Psorcon, the flurrenolone acetonide, Zpoflogin, amcinafal, Triamcinolone 16.alpha.,17.beta.-acetophenonide, Betamethasone Valerate and ester thereof, the Chloroprednisonum dragon, clorcortelone, the 21-descinolone, the terrestrial reference moral, dexamethasone, dichlorisone, difluprednate, flucloronide, fluorine compound, flunisolide, fluocinonide, fluocortolone, fluorometholone, fluperolone, fluprednisolone, meprednisone, the methyl meprednisone, paramethasone, prednisolone, prednisone.

Antibacterial reagent comprises sulfanilamide (SN), penicillin, cephalosporin, penicillinase, erythromycin, lincomycin, vancomycin, tsiklomitsin, paraxin, Streptomycin sulphate or the like.The example that the antimicrobial spy leads comprises erythromycin, carbonic acid ethyl erythromycin, propionic ester dodecyl erythromycin, gluceptate erythromycin, ethyl succinic acid erythromycin, erythromycin lactobionate, lincomycin, clindamycin, tetracycline, Uromycin, Demethylchlortetracycline, doxycycline, metacycline, oxidation tsiklomitsin, Minocycline HCl or the like.

Peptide and protein comprise, especially refer to the peptide littler than mean length, and for example Regular Insulin, vassopressin, pitocin, somatomedin, cytokine also have some big protein, for example human growth hormones.

Other reagent comprises various therapeutical agents, and is for example yellow fast, triamterene, theophylline, antineoplastic agent, 5 FU 5 fluorouracil nucleosides ribodesose, the Ismipur ribodesose, vidarabine, narcotic analgesic medicine, hydromorphone, cyclazine, analgesia is hot, and bupomorphine contains organic anion, heparin, prostaglandin(PG), the compound of the similar compound of prostaglandin(PG), FPL-670, carbenoxolone, poly oxy-compound, Dopamine HCL, Thomas's oxyphenisatin amine, the 1-Thomas, a-methyldopa, Angiotensin antagonistic, bradykinin for example, Regular Insulin, thyroliberin (ACTH), the brain coffee is barked, endorphin, Somatostatin, polypeptide that secretory product is such and for example tsiklomitsin, bromocriptine, lignocaine, the various mixing cpds of Cimitidine Type A/AB or any related compound.

Other reagent comprises iodate Brdurd, resin of podophyllium, theophylline, Racemic isoproterenol, Triamcinolone Acetonide phosphoric acid, hydrocortisone, indomethacin, Phenylbutazone, vitamin Bx and Trolovol.

Aforementionedly listedly exhausted do not include usedly, and all available the inventive method of any physiologically active reagent detects.

Typically, the database of structure is made up of the data of the quantitative generation of the demonstration by sequence specific PCR product.Typically, this database further contains the data that relate to serial correlation, gene mapping and cell distribution.

In one embodiment, the invention provides mRNA molecule 3 '-end and the identity of the sequence in the sequence library and the method for similarity that occurs in the sample that be identified in.It comprises the steps:

(a) mixture with an anchor primer prepares a double-stranded cDNA storehouse from a mRNA storehouse, and each anchor primer contains one 5 ' terminal and one 3 ' end, and comprises one section sequence of (i) 7-40T residue; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, first stuffer are positioned toward by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer inserts between the sequence of site that 6 above bases of identification are cut by first restriction enzyme and T residue; (v) from by-V ,-V-N and-each anchor primer phase transformation residue of selecting the group that V-N-N forms is positioned in 3 '-end, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N;

(b) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of second restriction enzyme identification has formed a double-stranded cDNA library of molecules that has first and second ends respectively;

(c) each the double-stranded cDNA molecule that derives from step (b) is inserted into into carrier in one direction, it is anti-meaning for the special promotor of phage, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', the 3 ' flanking vector that inserts cDNA sense strand 5 '-terminal and sense strand 3 '-end, and the said structure that one 3 ' flanking sequence arranged has 15 length of nucleotides in said first restriction endonuclease sites and qualification at least between transcription initiation site in described promotor;

(d) insert the carrier transformed host cell with the cDNA that is cut, contain the carrier that the clone inserts with generation;

(e) with at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the special promoter sequence of phage, but the structure storehouse that the digestion with restriction enzyme of sequence produces in step (c) on the energy identification carrier, so just produced the linear segment of the cDNA molecule that contains insertion, the linear fragment of Chan Shenging has a 5 ' flanking vector of holding at least 15 Nucleotide of second end of double-stranded cDNA molecule at carrier 5 ' like this;

(f) by incubation linear fragment and the special RNA polymerase of phage that has initial from the special promoter transcription ability of phage, produced the preparation of the cRNA that antisense cRNA transcribes;

(g) long with a ThermoScript II and a 15-30 Nucleotide, and, produce the first chain cDNA by transcribing cRNA by 5 ' the reverse transcription primer of forming with 5 ' flank body sequence complementary nucleotide sequence (RT);

(h) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA.The polymerase chain reaction is that 15-30 Nucleotide is long with first 3 '-PCR primer for the first time, it and determine 3 ' flanking vector sequence complementation between transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, first 5 ' PCR primer is defined to have-N ₁3 ' the end of forming, wherein " N " is among four kinds of thymus nucleic acid A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and 5 ' flanking vector preface complementation, simultaneously, the complementation of first 5 '-PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, in different primers of this first 5 ' PCR primer are used in four inferior storehouses of difference each;

(i) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product.Second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, it and determine 3 ' flanking vector sequence complementation between transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (h), x is from 1 to 5 integer, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, simultaneously complementary the extension with Nucleotide quantity of primer is equivalent to the special insertion Nucleotide that cRNA is inserted in " x "+1, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and has 4 for each inferior storehouse in the inferior storehouse of first cover in second series ^xYa Ku;

(j) analyze the second cover PCR product has produced the distinguished sequence of 3 ' of mRNAs that a representative occurs-terminal in the mRNA storehouse displaying;

(k) wash-out and corresponding at least one cDNA of mRNA that derives from the electrophoretogram, the band of 3 ' of the representative mRNAs that wherein occurs in sample-end is shown;

(l) the PCR product of amplification wash-out in a polymerase chain reaction;

(m) the isolating PCR product of clonal expansion enters in the plasmid;

(n) produce with the corresponding DNA of DNA that derives from plasmid clone;

(o) sequence of definite clone's cDNA;

(p) determine corresponding nucleotide sequence from a Nucleotide database, said here corresponding nucleotide sequence is begun the sequence that the place is defined by the recognition site of second restrictive restriction endonuclease least significant end and Poly (A) tail; And

(q) relatively clone cDNA sequence and corresponding nucleotide sequence, therefore can identify the 3 '-end sequence of the mRNA in the present sample and the identity and the similarity of a database sequence.

Typically, this method further comprises the steps:

(r) length and the quantity of comparison PCR product in a two-dimensional map shows;

Generally speaking, present method also comprises following a few step:

(s) determine corresponding Nucleotide prediction length (summation of the length of itself and the corresponding nucleotide sequence determined from database equates), can with the length of the length of 5 ' PCR sequence of carrier sequence hybridization, remaining anchor primer sequence, length that the carrier sequence gets involved part and with the length of interfertile 3 ' the PCR sequence of carrier sequence; With

(t) length of the corresponding nucleotide sequence of PCR product length and fixed prediction relatively is to be proved to be in the demonstration of a two-dimensional map by a graphic indicia or Chinese character in the length of the prediction of this corresponding nucleotide sequence.Suitable graphic indicia comprises the intersection short-term part of vertical and sea line, for example cruciform or " X " shape and comprises circular and polygonal geometricdrawing.

In another embodiment, the invention provides the method that is identified in identity and similarity between the corresponding cDNA fragment sequence of a mRNA molecule that sample occurs and sequence library, it comprises the steps:

Wash-out and the corresponding cDNA fragment of a mRNA molecule that in a sample, occurs;

The cDNA fragment of amplification wash-out produces the cDNA fragment of an amplification in the polymerase chain reaction;

The cDNA fragment of clonal expansion enters a plasmid;

Produce a dna molecular of and cDNA fragment correspondence;

Therefore the dna molecular that order-checking produces has determined the cDNA fragments sequence of wash-out; And,

Sequence in the cDNA fragments sequence of wash-out and the database relatively, identity and similarity that therefore can recognition sequence.

Typically, relatively the step of sequence is carried out with computer in the cDNA fragments sequence of wash-out and the database, and typically, present method also comprises the additional step that shows comparative result with diagram.

Generally speaking, the cDNA fragments sequence of a mRNA molecule correspondence and sequence identity and the similarity between database sequence occurring in a sample is to be undertaken by a method that comprises the steps:

Wash-out and the corresponding cDNA fragment of a mRNA that in a sample, occurs, the cDNA fragment has a length of being determined by the position of a Poly (A) tail of a restriction endonuclease sites and this mRNA molecule here;

With with one 5 ' PCR primer of this restriction endonuclease sites correspondence, determine the segmental partial sequence of cDNA by the polymerase chain reaction; And,

Compare the segmental definite partial sequence of wash-out cDNA and the segmental length of cDNA of the sequence in database, the identity and the similarity of coming recognition sequence.

In another embodiment, the invention provides and produce a method that transforms the registration of polynucleotide sequence database, it may further comprise the steps:

From a polynucleotide sequence database registration, select a source sequence;

A location poly (A) tail sequence in this source sequence;

One of location and the hithermost restriction enzyme enzyme recognition site of first recognition site sequence in source sequence;

Determine a index sequence of approximately forming by 2-6 Nucleotide with above-mentioned restriction endonuclease sites adjacency;

In source sequence, determine a relevant sequence, said correlated series comprises the sequence that is defined by poly (A) tail and restriction enzyme enzyme recognition site, and comprises at least a portion of restriction enzyme enzyme recognition site;

Determine the length of correlated series; With

Therefore storage can produce the database registration of a conversion about the information of the length of poly (A) tail position and sequence, correlated series that restriction enzyme enzyme recognition site position is relevant with source sequence with sequence.Typically, present method comprises that diagram shows the length of the correlated series relevant with index sequence.Preferably restriction endonuclease sites is from by MspI, selects in the group that TaqI and HinP1I form.

The present invention also provides by reducing because the background of non-target cDNAs amplification is improved a kind of method of the resolving power of the length of PCR product and quantity, and it comprises following step:

A sample in a cDNAs storehouse of screening, here each cDNA molecule includes insertion sequence and the sequence that derives from carrier;

Can carry out reverse transcription with the reverse transcription primer of the sequence hybridization that derives from carrier with one, extend about 5-6 Nucleotide and enter insertion sequence, to produce the product of a cDNA reverse transcription;

The product that separates the cDNA reverse transcription again;

Can carry out the polymerase chain reaction with one 5 ' primer at least one time with the product that separates the cDNA reverse transcription again with one 3 ' primer of the sequence hybridization that derives from carrier with one, extend about 7-9 Nucleotide and enter PCR product of insertion sequence generation, therefore reduce because the background of non-target cDNAs amplification.

The reverse transcription reaction reverse transcription primer different that 16 storehouses are typically arranged with 16.Always have 4 ^xThe polymerase chain reaction in inferior storehouse, here, the quantity that 5 ' PCR primer extension enters the Nucleotide of insertion sequence is different with the quantity that the reverse transcription primer extension enters the Nucleotide of insertion sequence.

These and other feature, aspect and advantage of the present invention will become with reference to following description, claims and the accompanying drawing that provides simultaneously and be more readily understood:

Fig. 1 is that diagram that the present invention improves one's methods is described, and it shows each stage of design of primers, cutting, clone, counter anticipate rna transcription and amplification, and diagram ground shown grappling and other primer sequence---complete sequence is referring to word segment;

Fig. 2 is described by the diagram of an embodiment who improves one's methods of the biotinylated anchor primer of the substrate of antibiosis protein chain mycin with having bag, and shown each stage of design of primers, cutting, clone, counter anticipate rna transcription and amplification, and diagram ground shown grappling and other primer sequence---complete sequence is referring to word segment;

Fig. 3 is with the relative abundance of the fluorescence detecting system mark PCR product chart to the relation of product length in base pair, it has shown the analysis to the PCR product that obtains with one 5 ' PCR primer C-G-A-C-G-G-T-A-T-C-G-G-G-G-T-G (SEQ ID NO:42), (A) begin from hungry serum (serun-starved) with the sample that adds the mRNA serum (serun-added) the people MG63 cell (B), with the software relative expression level between two samples relatively, be eclipsed at the end of sequence from (A) and the data that (B) obtain

Fig. 4 is for the method for two PCR steps with only use the method for a PCR step, use a fluorescence detecting system to compare the chart of the relative abundance of mark PCR product to product length in base pair, its show with or a PCR step (A-D) or two PCR steps (E-H) to (A) and add the mRNA that extracts serum (serun-added) the MG63 osteosarcoma cell (B) and analyze the result who obtains from hungry serum (serun-starved), shown and derived from 5 ' PCR primer 109T (C-G-A-C-G-G-T-A-T-C-G-G-T-G-C-A, SEO ID NO:43) and 45A (C-G-A-C-G-G-T-A-T-C-G-G-A-G-C-A, SEO ID NO:44) data, it is only in N1 position (representing with black matrix) difference, to hungry serum (serun-starved) (os-) and add serum (serun-added) (os ⁺) sample, showing that the PCR product that produces with 109T and 45A seems almost and be the same from the template that produces with a PCR step method (A-D), is diverse (E-H) and derive from the product that PCR detected that carries out next step with the template of the method generation of two PCR steps;

Fig. 5 is in order relatively to be used in the result that standard method described in Fig. 1 and the magnetic bead embodiment described in Fig. 2 are obtained, use a fluorescence detecting system to compare the chart of the relative abundance of mark PCR product to product length in base pair, it shows that the data of the embodiment that derives from magnetic bead and the data of the embodiment of the method that derives from standard compare, and are having a significant increase (producing the consistence of segmental similarity and strength values) aspect the replicability of all samples;

Fig. 6 is that expression is for the kurtosis of the PCR product of several different tissues generations and the figure of the linear dependence between the cDNA concentration;

Fig. 7 has shown nucleotide sequence and multiple clone site plasmid pBC SK ⁺/ DGT1, pBC SK ⁺/ DGT2, pBC SK ⁺/ DGT3, pBC SK ⁺/ DGT4 and pBC SK ⁺The restrictive collection of illustrative plates of/DGT5;

Fig. 8 is described by the diagram of an embodiment who improves one's methods of the biotinylated anchor primer of the substrate of antibiosis protein chain mycin with having bag; it has shown each stage of design of primers, cutting, clone, counter anticipate rna transcription and amplification, and diagram ground shown grappling and other primer sequence---complete sequence is referring to word segment.

We have researched and developed for the evaluation of sequence specific mRNAs synchronous in a mRNA storehouse and improving one's methods of demonstration, and this method is having a large amount of application aspect the screening of determining pharmaceutically-active mechanism, medicine, the research that reaches physiology and pathology situation, the gene mapping.Following is discussion to this improved method and application thereof.

The evaluation that the I.mRNAs synchronizing sequence is special

Based on polymerase chain reaction (PCR) technology; the method according to this invention provides the visible method that an obvious band is all arranged on gel by almost each mRNA of normal or neoplastic eukaryotic cell or tissue expression, and the concentration of the intensity of band and mRNA is roughly corresponding.Present method is based on containing 3 ' poly (A) tail, but does not rely in conjunction with on the basis of the observation of specific nearly all mRNAs of the primer of tail.

Improved method has proved as being illustrated in Fig. 1, Fig. 2 and three embodiment of Fig. 8.Generally speaking, improved method comprises following a few step:

A) mixture with an anchor primer prepares a double-stranded cDNA storehouse from a mRNA storehouse, and each anchor primer contains one 5 ' terminal and one 3 ' end, and comprises one section sequence of (i) 7-40T residue; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer are positioned toward by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer inserts 6 above bases of identification by between the sequence of first restriction enzyme inscribe cleavage site and T residue; (v) from by-V ,-V-N and-each anchor primer phase transformation residue of selecting the group that V-N-N forms is positioned in 3 '-end, preferred-V-N-N, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N;

(b) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of this second restriction enzyme identification is to form a double-stranded cDNA library of molecules that has first and second ends respectively;

(c) each the double-stranded cDNA molecule that derives from step (b) is inserted into into carrier, on direction, be anti-meaning for the special promotor of phage, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', the 3 ' flanking vector that inserts cDNA sense strand 5 '-terminal and sense strand 3 '-end, and the said structure that one 3 ' flanking sequence arranged has 15 length of nucleotides in said first restriction endonuclease sites and qualification at least between the transcription initiation site in described promotor;

(d) insert the carrier transformed host cell with the cDNA that is cut and produce the carrier that contains clone's insertion;

(e) with at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the special promoter sequence of phage, but the structure storehouse that the digestion with restriction enzyme of sequence produces in step (c) on the energy identification carrier, so just produced the linear segment of the cDNA molecule that contains insertion, the linear fragment of Chan Shenging has a 5 ' flanking vector sequence of holding at least 15 Nucleotide of second end of double-stranded cDNA molecule at carrier 5 ' like this;

(g) long with a ThermoScript II and a 15-30 Nucleotide, and, produce the first chain cDNA by transcribing cRNA by 5 ' the reverse transcription primer of forming with 5 ' flank body sequence complementary nucleotide sequence (RT):

(h) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA, this, polymerase chain reaction was that 15-30 Nucleotide is long with first 3 '-PCR primer first time, it and determine 3 ' flanking vector sequence complementation between transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, first 5 ' PCR primer has been defined-N ₁3 ' the end of forming, wherein " N " is among four kinds of thymus nucleic acid A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier preface complementation of 5 ' flank, simultaneously, the complementation of first 5 '-PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, is used in four inferior storehouses of difference each at different primers of this first 5 ' PCR primer;

(j) analyze the second cover PCR product has produced the distinguished sequence of 3 ' of mRNAs that a representative occurs-terminal in the mRNA storehouse displaying.

In an optional embodiment, above-mentioned steps (c) comprises that inserting each the double-stranded cDNA molecule that derives from step (b) enters carrier, and the direction of its insertion is significant to the special promotor of carrier pnagus medius, has so just formed one and has contained the structure storehouse (Fig. 8) of inserting the cDNA molecule.

A. the preparation of double-stranded cDNA

The first step in the method requires a mRNA storehouse.The method of extracting mRNA is known and is described in the field hereto.For example, at J.Samrook etc., " molecular cloning: experiment guide " (Cold Spring Harbor Laboratory Press, Cold Spring Habor, NewYork, 1989), vol.1, ch.7, " extraction from eukaryotic cell, purifying and analysis messenger RNA(mRNA) ", at this by incorporated by reference.Other separation and extracting method also are known.At large, separation is to carry out under the situation that resembles the existence of chlorination guanidine or guanidine thiocyanate chaotropic agent, although other stain remover and extraction agent also are available.

Usually, mRNA be by oligomerization dT-cellulose chromatography or other have the ability separate from total extraction RNA in conjunction with the chromatographic media of 3 '-part of the polyadenylic acidization of mRNA molecule.What can select is that total RNA is an available, but is not most preferred.But, generally speaking extract poly (A) ⁺RNA is preferable.

With the reverse transcription of the initial initiation of the mixture of an anchor primer, so just prepared double-stranded cDNAs from the mRNA storehouse.Each anchor primer contains one 5 ' terminal and one 3 ' end, and comprises one section sequence of (i) 7-40T residue; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer are positioned toward by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer inserts 6 above bases of identification by between the sequence of first restriction enzyme cleavage site and T residue; (v) from by-V ,-V-N and-each anchor primer phase transformation residue of selecting the group that V-N-N forms is positioned in 3 '-end, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N.Here the phase transformation residue in the mixture by-V ,-V-N or-among the V-N-N one limit.Anchor primer has the phase transformation residue of one-V here, and mixture comprises the mixing of 3 anchor primers.Anchor primer has the phase transformation residue of one-V-N here, and mixture comprises the mixing of 12 anchor primers.Anchor primer has the phase transformation residue of one-V-N-N here, and mixture comprises the mixing of 48 anchor primers.

Be typically, each anchor primer has 18 T residues in T residue sequence zone, at-V-N-N end first long stuffer of 14 residues is arranged, the preferred sequence of first stuffer is to screen from the group that is made up of A-A-C-T-G-G-A-A-G-A-A-T-T-C (SEQ ID NO:1) and G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ IDNO:2).Representative is that the cleavage site of the restriction enzyme of 6 above bases of identification is NotI cleavage sites.

3 can have A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ IDNO:3) sequence by a preferred cover anchor primer.12 can have A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (S EQ ID NO:4) sequence by a preferred cover anchor primer.48 can have A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (S EQ ID NO:5) sequence by a preferred cover anchor primer.

In a preferred embodiment, 3 anchor primers of that cover have the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:6).In another preferred embodiment, 12 anchor primers of that cover have the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO:7).In another particularly preferred embodiment, 48 anchor primers of that cover have the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:8).

Therefore synthetic in a 3 ' terminal fixed position of all copies of an initial initiation of member each mRNA species in sample of described anchor primer mixture define one 3 ' distal point of each species.

This reaction is to carry out under the condition of carrying out double-stranded cDNA preparation from mRNA, and this people for this area is known.For example such technology in " molecular cloning: experiment guide " the 2nd volume, has been described in the title " structure in cDNA library and analysis " at J.Samrook etc.Suitable ThermoScript II comprises the ThermoScript II of the leukosis virus (MMLV) that derives from bird myeloblastemia syndrome virus (AMV) and Moloney mouse.One preferably ThermoScript II be the MMLV ThermoScript II.

In a preferred embodiment of the invention, magnetic bead is used to improve the preparation (Fig. 2 and Fig. 8) in cDNA storehouse.Representative is, the part of vitamin H is the 5 ' end that conjugation is connected to anchor primer, and by being contacted first restrictive cDNA by the substrate of antibiosis protein chain mycin with bag, first restrictive cDNA is separated from remaining cDNA, as wrapping by the quantity of antibiosis protein chain mycin magnetic bead.

B. with restriction enzyme cutting cDNA sample

The cDNA sample cuts with two kinds of restriction enzymes.The site that first restriction enzyme identification has 6 above bases, single site cutting in each member of anchor primer mixture simultaneously.Second restriction enzyme is the restriction endonuclease that can discern a 4-nucleotide sequence.Such restriction endonuclease generally cuts at the multidigit point of most cDNAs.Representative is, first restriction enzyme is NotI, and second restriction enzyme is MspI.NotI can not cut in most of cDNAs.We are desirable just for this, and it has reduced because the loss of inserting the clone who causes except the cutting at the cDNAs of anchor station external position.

Second restriction endonuclease sites can be at TaqI, MaeII, or select among the HinP1I.Can detect the rare mRNA s that can not be cut by MspI with three above-mentioned restriction enzymes.Second restriction enzyme produce one 5 '-outstanding, as following described, its matches with the expectation carrier of cloning.This clone is from by pBC SK for carrier ⁺, pBS SK ⁺, pBCSK ⁺/ DGT1, pBS SK ⁺/ DGT2 and pBS SK ⁺Screen in the group that/DGT3 forms, the clone enters the ClaI site, resembles as discussed below.

Second restriction enzyme can be selected Sau3AI.Each and every one restriction enzyme can detect the rare mRNA s that can not be cut by MspI with this.Second restriction enzyme produce one 5 '-outstanding, as below discussed, it matches with the expectation carrier of cloning.This cloning vector is pBS SK ⁺/ DGT4, the clone enters the BamHI site, as below discussed.

Second restriction enzyme can be selected NlaIII.Each and every one restriction enzyme can detect the rare mRNA s that can not be cut by MspI with this.Second restriction enzyme produce one 5 '-outstanding, as below discussed, it matches with the expectation carrier of cloning.This cloning vector is pBS SK ⁺/ DGT5, the clone enters the SphI site, as below discussed.

Can be used to detect and to be selected by other the restriction enzyme that is fit to of the cDNAs of above-mentioned restriction enzyme cutting.The suitable restriction enzyme that can discern a 4-nucleotide sequence is MboI, DpnII, Sau3AI, Tsp509I, HpaII, BfaI, Csp6I, MseI, HhaI, NlaIII, TaqI, MspI, MaeII and HinP1I.

First restriction enzyme that is fit to of 6 above bases of identification is AscI, BaeI, FseI, NotI, PacI, PmeI, PpuMI, RsrII, SapI, SexAI, SfiI, SgfI, SgrAI, SrgfI, Sse8387I and SwaI.

The condition of digested cdna is known in this field, for example at J.Samrook etc., in " molecular cloning: experiment guide " the 1st volume the 5th chapter, has been described in " being used in the enzyme in the molecular cloning ".

C. the cDNA of cutting injects in the carrier

Then, the cDNA sample of first restriction enzyme and second restriction enzyme cutting injects in the carrier, summarizes and says that suitable carriers contains the multiple clone site of a NotI restriction endonuclease sites.Plasmid pBC SK with restriction enzyme ClaI and NotI cutting ⁺It is a suitable carriers.This carrier contains the special promotor of phage.Representative is that promotor is T3, a SP6 or T7 promotor.A preferred promotor is the promotor of phage T3.It is in the promotor on the direction of anti-meaning (Fig. 1 and Fig. 2) that the cDNA of cutting is inserted in the promotor special with respect to phage.In another embodiment, to be inserted in the promotor special with respect to phage be in the promotor on the significant direction (Fig. 8) to the cDNA of cutting.In a preferred embodiment, contain the carrier of a multiple clone site by a nucleotide sequence that from the group that forms by SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, screens.

Based on plasmid vector pBluescript (pBS or pBC) SK ⁺(Stratagene) carrier is preferred, and this plasmid vector is deleted in the nucleotide sequence part of from 656 to 764 positions, replaces with at least 110 nucleotide sequences that include a NotI restriction endonuclease sites.The nucleotide sequence part from the SacI site to the KpnI site has been crossed in this zone that is designated as multiple clone site (MCS).

A suitable plasmid vector, for example pBC SK ⁺Or pBS SK ⁺(Stratagene), with at least 100 Nucleotide of suitable digestion with restriction enzyme with the deletion multiple clone site.For pBS SK ⁺, the restriction enzyme that is fit to of deletion multiple clone site is SacI and KpnI.Form by a new multiple clone site, contain after and can partly clone into that this carrier forms a suitable plasmid vector with a cDNA of the end of NotI and ClaI coupling with first and second digestion with restriction enzyme.The preferably cDNA that is made up of a new multiple clone site partly contains the part of describing in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.Be listed in the interior single digestion with restriction enzyme with 6 sequences more than the base by discerning the 4-nucleotides sequence that comprises second restriction endonuclease sites with one, cDNA clones by linearization.

A preferred plasmid vector refers to pBC SK here ⁺/ DGT1, it comprises the multiple clone site (MCS) of SEQ IDNO:9.Second restriction enzyme and linearization restriction enzyme to (step e, below) are respectively: MspI and SmaI; HinP1I and NarI; TaqI and XhoI; MaeI1 and AatII.

A preferred plasmid vector refers to pBS SK here ⁺/ DGT2, it comprises the multiple clone site (MCS) of SEQ IDNO:10.And as above-mentioned pBC SK ⁺/ DGT1 equally prepares.The cDNA that this multiple clone site is not accepted to produce with MaeI1 inserts.Therefore, for pBSSK ⁺/ DGT2, second restriction enzyme and linearization restriction enzyme to (step e, below) are respectively: MspI and SmaI; HinP1I and NarI; TaqI and XhoI.

Another preferred plasmid vector refers to pBS SK here ⁺/ DGT3, it comprises the multiple clone site (MCS) of SEQ IDNO:11.Second restriction enzyme and linearization restriction enzyme to (step e, below) are respectively: MspI and SmaI; HinP1I and NarI; TaqI and XhoI; MaeI1 and AatII.

Another is plasmid vector preferably, refers to pBC SK here ⁺/ DGT4, it comprises the multiple clone site (MCS) of SEQID NO:12.Being suitable for second restriction enzyme of this carrier and linearization restriction enzyme to (step e, below), is respectively Sau3AI and BglII.

Another is plasmid vector preferably, refers to pBS SK here ⁺/ DGT5, it comprises the multiple clone site (MCS) of SEQ IDNO:13.Be suitable for second restriction enzyme of this carrier and linearization restriction enzyme to (step e, below), NlaIII and NcoI respectively.

In a preferred embodiment, carrier contains a carrier padding sequence, and this carrier padding sequence comprises that an interior carrier between first and second carrier restriction endonuclease sites fills restriction endonuclease sites.In a such embodiment, linearization step comprises that filling the restriction enzyme that restriction endonuclease sites can cut vector with one in interior carrier digests carrier.In another embodiment, the restriction enzyme that is used in the linearization step also can cut the carrier of filling restriction endonuclease sites in interior carrier.

D. the conversion of a proper host cell

The carrier that the DNA of cutting has inserted then is used to transform a suitable host cell, and this host cell is efficiently transformed or transfection by containing the segmental carrier of insertion.For clone's proper host cell, resemble Samrook etc., " molecular cloning: experiment guide ", most representative host cell is a protokaryon.A particularly suitable host cell is that a colibacillary strain is.A suitable colibacillary strain is to be MC1061.More preferably, little equal portions are used to the XL1-Blue strain system of transformed into escherichia coli, so that by determine to insert clone's percentage at the relative percentage ratio of the dull and stereotyped blueing hickie of X-gal.Have only the library to have above 5x10 ⁵Reorganization is just generally accepted.

The segmental generation of linearization of E

Then from each cDNA library, prepare plasmid.Produce linearization fragment by at least a digestion with restriction enzyme.

In one embodiment, carrier is plasmid pBC SK ⁺, and NspI not only is used to second restriction enzyme but also is used to linearization restriction enzyme.

In another embodiment, carrier is plasmid pBC SK ⁺, second restriction enzyme be from by: screen the group that MspI, MaeII, TaqI and Hin1P form.Linearization is by first digestion with SmaI, and second digestion with KpnI and ApaI mixture is subsequently finished.

Carrier is from by pBC SK in another embodiment ⁺/ DGT1, pBS SK ⁺/ DGT2, pBSSK ⁺/ DGT3, pBC SK ⁺/ DGT4 and pBS SK ⁺Select in the group that/DGT5 forms.In this embodiment, providing the combination of a suitable enzyme, is MspI at this second restriction enzyme, and the enzyme that is used in linearization is SmaI.The combination of the enzyme that is fit to that another provides is that second restriction enzyme is TaqI, and the enzyme that is used in linearization is XhoI.The combination of the enzyme that is fit to that further provides is that second restriction enzyme is HinP1I, and the enzyme that is used in linearization is NarI.The combination of the enzyme that is fit to that another provides is that second restriction enzyme is MaeII, and the enzyme that is used in linearization is AatII.If carrier is pBC SK ⁺/ DGT4, the combination of the enzyme that is fit to that provides is that second restriction enzyme is Sau3AI, the restriction enzyme that is used in the linearization step is Bg1II.If carrier is pBS SK ⁺/ DGT5, the combination of the enzyme that is fit to that provides is that second restriction enzyme is NlaIII, the restriction enzyme that is used in the linearization step is NcoI.

Generalized theory, below in the linearization step that F partly describes in detail, the plasmid vector that cDNA of any shortage inserts all is cut at the recognition site place (at the underscore place of Fig. 7 A) of a 6-Nucleotide, find it is between NotI and ClaI site for SmaI, NarI, XhoI or AatII, and find 3 ' near the ClaI site for 6 the above recognition sites of base that have of SmaI, NarI, XhoI or AatII.Contrast the plasmid vector finding to contain insertion sequence for SmaI, NarI, XhoI, or the AatIII site have 6 nucleotide sequence recognition sites 3 ' near the ClaI site.

The generation of F.cRNA

Next step is the generation of antisense cRNA cRNA preparation of transcribing, and this is to have an initial RNA polymerase that derives from the special promoter transcription of phage by the linearizing fragment of incubation and one to carry out.Representative ground, just as discussed above, promotor is a T3 promotor, so polysaccharase is the T3RNA polysaccharase.Under suitable synthesis condition (Ambion, Austin, TX), polysaccharase and linearizing fragment and 4 triphosphoric acid Yeast Nucleic Acid incubations.

G. transcribing of the first chain cDNA:

(life technology, Gaithersburg MD) transcribe the first chain cDNA with sick (MMLV) institute transcriptase whenever of the leukemia of Moloney mouse.This ThermoScript II is 42 ℃ of annealing down, and responsive transcription carries out under 42 ℃.The primer of a 15-30 length of nucleotides is used in this reaction, and this primer and 5 ' flanking vector sequence complementation.

In another embodiment, transcribe cRNA with a heat-staple ThermoScript II, and primer as described below, the recombinant retroviruses enzyme that preferred ThermoScript II is a bird, it is called as thermally-stabilised ThermoScript II of transcribing (ThermoScript RT), and it is applicatory recording and narrating according to life technology (GaithersburgMD).

This has improved complementarity highly loyal between between primer and cRNA.At least 15 length of nucleotides of this primer, and the sequence of the promotor 3 ' special with phage-end is corresponding.

Another ThermoScript II that is fit to is the recombinant retroviruses enzyme that derives from the Thermus thermophile bacteria, and it is called as rTh, and (Norwalk, CT) report is an available according to Perkin-Elmer.

Here the special promotor of phage is the T3 promotor, and primer typically has the sequence of tool A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO:14) or G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:47).

H. produce first PCR product

Next step is to use the product of transcribing as template, carries out the fragment of the amplification of generation polymerase chain reaction, polymerase chain reaction with the first cover primer as described below.

Put it briefly, the product that the first chain cDNA transcribes is used as template, carries out the polymerase chain reaction with one first 3 ' PCR primer and 5 ' PCR primer that overlaps, and produces the amplified fragments of polymerase chain reaction.First 3 ' PCR primer 15-30 Nucleotide typically is long, and coexist first restriction endonuclease sites and the site that limited by the special promotor of phage between 3 ' flanking vector sequence complementation.First 5 ' PCR primer has one by N ₁One 3 ' end, N here of group ₁Be among 4 deoxyribonucleotide A, C, G or the T, and this primer is that 15-30 Nucleotide is long, itself and 5 ' flanking vector sequence complementation, because the complementarity of primer, it extends into a Nucleotide in the Nucleotide of special insertion of cRNA.Used a different primer in first 5 ' PCR primer in 4 different inferior storehouses each.

When carrier is the plasmid pBC SK that cuts with ClaI and NotI ⁺The time, a 3 ' suitable PCR primer is to select from the group that is made up of G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:47) and G-A-G-C-T-C-G-T-T-T-T-C-C-C-A-G (SEQ ID NO:48).Here the special promotor of phage is the T3 promotor, and suitable 5 '-PCR primer has the sequence of G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:22), is A, G, C or T among the N in a given reaction wherein.

Typically, with a PCR program, PCR is 94 ℃ of sex change 15 seconds, annealed 15 seconds for 50 ℃-65 ℃, under 72 ℃ of synthetic 30 seconds conditions, (Pekin-Elmer Cetus, Norwalk CT) carry out on such thermal cycler at a suitable for example PTC-200 (MJ Research) or Pekin-Elmer 9600.Specific nucleotide sequence annealing temperature for primer is optimised, and its used principle is known to this field.High-temperature annealing step is that mispairing minimizes by making the people at its 3 ' first terminal 5 '-PCR primer, can promote highly loyal duplicating simultaneously.

I. produce second PCR product

Next step is to make template with the product of first PCR reaction, uses one second cover primer as described below to carry out the polymerase chain reaction second time, produces the amplified fragments of the second cover polymerase chain reaction.

Put it briefly, as template, utilize one second 3 ' PCR primer and second 5 ' PCR primer to carry out the polymerase chain reaction, produce the polymerase chain reaction (PCR) amplification fragment with first PCR reaction product.Typically, this second 3 ' PCR primer is that 15-30 Nucleotide is long, it and determine that at first restriction endonuclease sites with by the special promotor of phage 3 ' flanking vector sequence complementation between transcription initiation site, this second 5 ' PCR primer have been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (h), x is from 1 to 5 integer, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, simultaneously complementary the extension with Nucleotide quantity of primer is equivalent to the special insertion Nucleotide that cRNA is inserted in " x "+1, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and has 4 for each inferior storehouse in the inferior storehouse of first cover in second series ^xYa Ku.

In another embodiment, used primer is: the sequence in insertion site that abuts against the cDNA sample in the carrier in the carrier that (a) coexists corresponding one second 3 ' PCR primer on sequence; (b) first 5 ' PCR primer that from be used in first PCR reaction by (i), carries out the amplification of inferior storehouse; (ii) deriving from the first chain cDNA is used to by an additional-N residue 3 ' terminal first 5 ' PCR primer that extends at Ya Ku; (iii) be used in inferior storehouse 3 '-first 5 ' PCR primer that end extends by two additional-N-N residues; (iv) be used at inferior storehouse 3 ' terminal first 5 ' PCR primer that extends by three additional-N-N-N residues; (v) being used to select in the group of composition at inferior storehouse 3 ' terminal first 5 ' PCR primer that extends by four additional-N-N-N-N residues, can be among A, C, G or the T any at this N.

3 ' the PCR primer that is fit to is to screen from the group that is made up of G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ IDNO:47) and G-A-G-C-T-C-G-T-T-T-T-C-C-C-A-G (SEQ ID NO:48).

Here the special promotor of phage is the T3 promotor, and suitable 5 '-PCR primer is to select from the group that is made up of following sequence:

A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N(SEQ?ID?NO：16)；

A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N(SEQ?ID?NO：17)；

A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N(SEQ?ID?NO：18)；

G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N(SEQ?ID?NO：22)；

G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N(SEQ?ID?NO：23)；

T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N(SEQ?ID?NO：24)；

C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N(SEQ?ID?NO：25)；

G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N(SEQ?ID?NO：26)；

A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N(SEQ?ID?NO：16)；

A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO:19); And

A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N(SEQ?ID?NO：20)。

Typically, with a PCR program, PCR is 94 ℃ of sex change 15 seconds, annealed 15 seconds for 50 ℃-65 ℃, under 72 ℃ of synthetic 30 seconds conditions, (Pekin-Elmer Cetus, Norwalk CT) carry out on such thermal cycler at a suitable for example PTC-200 (MJ Research) or Pekin-Elmer 9600.Specific nucleotide sequence annealing temperature for primer is optimised, and used principle is known to this field.The high-temperature annealing steps is that mispairing minimizes by making the people at its 3 ' first terminal 5 '-PCR primer, promotes highly loyal duplicating simultaneously.

But the detection method of utilizing nonradioactive labeling's preferred embodiment is an available.For the on-radiation detection method, preferably one of primer that reacts for second PCR is by fluorescent mark of conjugated.Suitable fluorescent mark is from by selecting the group that forms below.

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, the 6-carboxylic acid, 3 ', 6 '-dihydroxyl-6-Fluoresceincarboxylic acid (6-FAM, ABI);

9-(2,5-dicarboxyl phenyl)-3,6-two (dimethylamino)-xanthene (6-carboxyl tetramethylrhodamin (6-TAMRA), molecular probe);

3, (2-carboxyl phenyl)-(rhodamine is green for xanthene for 6-diamino-9- ^TM, molecular probe);

1H, 5H, 11H, 15H-xanthene o[2,3,4-ij:5,6,7-i ' j '] two quinolizines-8-,-(2,4-two sulphur benzene)-2,3,6,7,12,13,16,17-octahydro one, inner salt (Texas Red, molecular probe);

6-((4,4-two fluoro-1,3-dimethyl-5-(4-p-methoxy-phenyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino)-caproic acid (BODIPY TMR-X, molecular probe);

6-(((4-(4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) benzene oxygen) acetyl) amino)-caproic acid (BODIPY TR-X, molecular probe);

4,4-two fluoro-5-styryl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid;

6-(((4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) styryl oxygen) ethanoyl) hexosamine (BODIPY 630/650, molecular probe);

9-(2,4 (or 2,5)-dicarboxyl phenyl)-3,6-two (dimethylamino)-xanthene, inner salt (TAMRA, molecular probe);

Other fluorescent mark applicatory comprises 4,7,2 ', 4 ', 5 ', 7 ' chlordene-6-Fluoresceincarboxylic acid (" HEX ", ABI) and 4,7,2 ', 7 ' tetrachloro-6-Fluoresceincarboxylic acid (" TET ", ABI) and " NED " (ABI), it is that this field is known.

One preferably the PCR mark be spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 6-carboxylic acid, 3 ', 6 '-dihydroxyl-6-Fluoresceincarboxylic acid (6-FAM).

In an optional embodiment, the detection method of available reflection autography.In one embodiment, PCR be ³⁵S-dATP carries out under existing.Selectively, pcr amplification can be at the deoxyribonucleoside triphosphate of a radioisotope labeling, for example [ ³²P] dCTP or [ ³³P] dCTP carries out under existing.Yet in order to obtain maximum resolving power, radioactive automatic developing detects usually preferred with one ³⁵The deoxyribonucleoside triphosphate of S-mark.

In an optional embodiment, with the oligonucleotide detection method of magnetic grain mark just as at U.S. Patent number: 5,656, equally can be employed and detect described in 429, this method at this by incorporated by reference.

In a preferred embodiment, connect by the thiophosphatephosphorothioate ligase enzyme at 3 ' of first or second 5 ' PCR primer 3 terminal Nucleotide.Referring to Mullins, J.I., de.Noronha, C.M with 3 '-degraded of the terminal anti-crater of thiophosphatephosphorothioate ligase enzyme amplimer polysaccharase, and can reduce the Taq polysaccharase and misquote and lead.PCR method uses 1,992 2 (2): 131-136; Ott, J. and Eckstein, F, the anti-dna polymerase i degraded of protection oligonucleotide primer.Biological chemistry 1,987 26 (25): 8237-8241; Uhlmann, E., Ryte, A., and Peyman, the stable Mechanism Study of the anti-karyolysis degraded of A. part thiophosphatephosphorothioate oligonucleotide.Anti-meaning nucleic acid drug progress, 1,997 7 (4): 345-350; Schreiber, G.K, Koch, E.M., and Neubert, W.J. is by the combination of the similar thing of thiophosphatephosphorothioate, the selective protection of external synthetic cDNA nuclease-resistant, nucleic acids research 1,985 13 (21): 7663-7672.

J. electrophoresis

The fragment of polymerase chain reaction (PCR) amplification then goes to show that by for example electrophoretic separation method representative appears at the 3 '-terminal band of the mRNAs in the sample, can be come out by explanation.

The electrophoretic technique of resolved analysis pcr amplified fragment is thoroughly understood in this field, here there is no need to be described in further detail.Corresponding PCR product is analyzed by explanation on the denatured DNA sequencing gel, and is visible by the laser apparatus of induced fluorescence.Alternatively, corresponding PCR product can be analyzed with capillary electrophoresis explanation, and is visible by the laser apparatus of induced fluorescence.

In a preferred embodiment, there is one to be connected a fluorescent mark in the primer of second PCR reaction by conjugation.Suitable fluorescent mark is from by selecting following the composition the group:

9-(2,5-dicarboxyl benzene)-3,6-two (dimethylamino)-xanthene (6-carboxyl tetramethylrhodamin (6-TAMRA), molecular probe);

3, (2-carboxyl benzene)-(rhodamine is green for xanthene for 6-diamino-9- ^TM, molecular probe);

1H, 5H, 11H, 15H-xanthene o[2,3,4-ij:5,6,7-i ' j '] two quinolizines-8-,-(2,4-two sulphur benzene)-2.3,6,7,12,13,16,17-octahydro, inner salt (Texas Red, molecular probe);

6-((4-4-two fluoro-5,7---methyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino) caproic acid (BODIPX FL-X, molecular probe);

9-(2,4 (or 2,5)-dicarboxyl benzene)-3,6-two (dimethylamino)-xanthene, inner salt (TAMRA, molecular probe).

Other fluorescent mark applicatory comprises 4,7,2 ', 4 ', and (" HEX ", ABI), " NED " (ABI) with 4,7, (" TET ", ABI), it is that this field is known to 2 ', 7 ' tetrachloro-6-Fluoresceincarboxylic acid to 5 ', 7 ' chlordene-6-Fluoresceincarboxylic acid.

Typically, fluorescence is used to detect the cDNA species of distinguishable analysis.Yet it also is available that other detection method resembles phosphorescent substance imaging or radioautograph or magnetic detection.

According to scheme, contain nearly all poly (A) in initial RNA sample in the cDNA library that from each mRNA sample, produces ⁺3 '-farthest terminal copy that begins to poly (A) tail from the MspI site of farthest side of mRNAs, the relative concentration of it and initial mRNAs almost is corresponding.Because two ends of each species insertion sequence are all by the accurate qualification of sequence institute, so for each species, their length is consistent, and this makes them that a concrete visible band be arranged on gel, and no matter mRNA how tissue-derived.

Typically, the product intensity that shows behind the electrophoresis is approximately proportional with the abundance of the mRNAs that corresponds to the product in the original stock.

Typically, present method comprises further behind the electrophoresis that the intensity that corresponds to mRNA by product determines a step of the relative abundance of each mRNA in original stock.

II. present method is to the application of mRNA demonstration

Pattern

The analysis of the detection of mRNA expression pattern and these patterns had a large amount of application during present method of foregoing description was organized by gel electrophoresis.During these are used one is the application that detects the relation that the variation of mRNA expression pattern in tissue and physiology or pathology change.

Summarize and say that present method comprises:

(1) acquisition is not subjected to the sample of physiology or first tissue of pathology variation;

(2) as mentioned above, by carrying out and represent the method for the special evaluation of synchronizing sequence of the corresponding mRMAs of member in an anti-meaning cRNA storehouse of 3 ' of a mRNAs storehouse-end, determine mRNA expression pattern in first sample of tissue, produce the demonstration that representative appears at first band of 3 '-end of mRNA in first sample;

(3) acquisition is subjected to the sample of second tissue of physiology or pathology variation;

(4) as mentioned above, by carrying out and represent the method for the special evaluation of synchronizing sequence of the corresponding mRMAs of member in an anti-meaning cRNA storehouse of the 3-end in a mRNAs storehouse to determine mRNA expression pattern in second sample of tissue, produce the demonstration that representative appears at first band of 3 ' of mRNA in second sample-terminal;

(5) relatively first and second demonstrations determine to change on physiology or the pathology influence to mRNA expression pattern in the tissue.

Typically, relatively be in the swimming lane of the adjacency of a single gel, to carry out.

Typically, comprise the database that the quantitative generation data of the demonstration by the sequence specific product are formed,, constructed in hardware soft and keeping with suitable computer.More preferably, such database further comprises the data of, gene mapping relevant about sequence and cell distribution.In a preferred embodiment, the numerical value of length and the prediction determined with a database that derives from nucleotide sequence of the part of the nucleotide sequence of PCR product is at least compared.

Tissue can be to derive from central nervous system, especially, it can derive from central nervous system, and it is retina, cerebellar cortex, sense of smell bubble, thalamus, hypothalamus, preceding hypophysis, back hypophysis, hippocampus (hippocampus), nucleus accumbens septi, tonsilla, striatum, cerebellum, brain stem, suprachiasmatic nucleus or backbone rope.In central nervous system, physiology or pathological variation can be following senile dementia, Parkinson's neurological dysfunction, local asphyxia, be addicted to drink, be addicted to drug, in the schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, dysthymia disorders, bipolar manic depressive disorder any when tissue-derived.Alternatively, method of the present invention can be used to studying physiological tempo variation, aging and long-term the reinforcement, and the latter influences same hippocampus.Alternatively, especially with appear at central nervous system in the relevant mRNA species of ad hoc structure, present method can be used to study the brain region of the participation complex behavior that oneself knows, for example learn, memory, mood, be addicted to drug, the glutamate dehydrogenase neurotoxic, feed behavior, sense of smell, virus infection, vision and moving obstacle.

Present method can be used to study the result of medicine and/or toxin dispensing by mRNA pattern in tissue of comparative drug or toxin dispensing front and back.The result of electro-shock therapy method also can be studied.

Selectively, tissue-derivedly comprise cardiovascular systems, pulmonary system, Digestive tract, peripheral nervous system, liver, kidney or skeletal muscle and reproductive system or derive from any other organ or the tract of health in a tissue or the system of organ.For example, the mRNA pattern can be used to research and derive from liver, the heart, kidney or skeletal muscle.In addition, for any tissue, sample can extract so that find the circadian influence that mRNA expresses in each time.Like this, present method is attributable to specific mRNA species and has participated in specific function or dysfunction pattern.

More preferably, normal or neoplastic tissue comprises that organ of screening from the group that is made up of neural system, endocrine system, body (being comprised skin, hair and nail), Skeletal system (comprising bone and muscle), urinary system and the reproductive system of cardiovascular systems, lymphsystem, respiratory system, Digestive tract, peripheral nervous system, central nervous system, intestines or organ-tissue extract or the deutero-cell.

In a preferred embodiment, the normal or neoplastic tissue of being made up of cell comprises by from epithelium, endothelium, mucous membrane, body of gland, blood, lymph, conjunctive tissue, cartilage, bone, unstriated muscle, skeletal muscle, cardiac muscle, neurone, neurogliocyte, spleen, thymus gland, hypophysis, Tiroidina, parathyroid gland, adrenal cortex, adrenal medulla, pineal gland, skin, hair, nail, tooth, liver, the pancreas lung, kidney, bladder, ureter, mammary gland, ovary, the uterus, vagina, testis, prostate gland, penis, organ that screens in the group of an E ﹠ E composition or organ-tissue extract or the deutero-cell.

Similarly, mRNA analytical procedure of the present invention can be used as the part of screening of medicaments side effect method, and generally speaking, this quadrat method comprises:

(1) from obtains first duplicate samples of tissue with the organism of the compound treatment of known physiologic function;

(2) as mentioned above, by carrying out and represent the method for the special evaluation of synchronizing sequence of the corresponding mRMAs of member in an anti-meaning cRNA storehouse of 3 ' of a mRNAs storehouse-end to determine mRNA expression pattern in first sample of tissue, produce the demonstration that representative appears at first band of 3 ' of mRNA in first sample-terminal;

(3) from obtain second duplicate samples of tissue with the organism of the drug treating of screened side effect;

(4) as mentioned above, by carrying out and represent the method for the special evaluation of synchronizing sequence of the corresponding mRMAs of member in an anti-meaning cRNA storehouse of a mRNAs storehouse 3 '-end to determine mRNA expression pattern in second sample of tissue, produce the demonstration that representative appears at second band of 3 ' of mRNA in second sample-terminal;

(5) compare first and second demonstration, so that detect the existence of mRNA species, the expression of mRNA is not subjected to the influence of known compound, but be subjected to the influence of screened medicine, therefore, show the difference of the effect of the medicine of screening and known compound, thereby side effect is arranged.

Especially, present method can be used to influence the medicine of central nervous system, for example by thymoleptic, Antipsychotic drug, tranquilizer, anticonvulsive drug, oxidase inhibitor, stimulant.Yet this method can be used to influence the screening of arbitrary medicine that mRNA expresses in a specific tissue in practice.For example can study anti-Parkinson agent, skeletal muscle relaxant, anodyne, toponarcosis, cholinergic agent, spasmolytic, steroid and non-steroid anti-inflammatory drug, antiviral agent or any other and have and influence the influence that medicine that mRNA expresses is expressed mRNA, and determine the influence in a particular organization or structure.

The further application of the inventive method is the 3 '-terminal sequence that obtains the mRNA species be shown.Summarize and say, the method that obtains sequence may further comprise the steps:

(1) cDNA of at least one and a mRNA correspondence of wash-out from electrophoretogram, the 3 '-terminal band that representative appears at the mRNA in the sample in this electrophoretogram is shown;

(2) in a polymerase chain reaction, eluted cDNA increases;

(3) cDNA of clonal expansion enters a plasmid;

(4) DNA of the DNA correspondence that from plasmid, produces and clone; And

(5) order-checking clone's cDNA.

Be used in the cDNA that second primer amplification in the PCR step has been sheared with the front.This cDNA then by the TA clone be connected enter carrier and by the clone advance pCRII (Invitrogen, SanDiego, CA).Then from subclone, prepare DNA in a small amount, and part is carried out automatic sequencing by the double deoxidating chain end method of Sanger by sex change be divided into two equal portions with standard technique.Available for example ABI sequenator commercial suitable sequenator like this checks order.This will allow the mensuration of length range at most of cDNA complementary sequences of the research of 50-500bp.Then with suitable computer configuration, these partial sequences can be used to scan the few nucleotide of GenBank for example according to base, and usefulness is the such comparison of BLASTN and BLASTX and the identity and the similarity of routine analyzer recognition sequence for example.Because this method produces the sequence of the 3 ' end that only derives from mRNA, expectation open reading frame (ORFs) is only run into occasionally.For example, 3 ' of brain-untranslated district mean length be longer than 1300 Nucleotide (J.G.Sutcliffe, 1988, supra).By label protein motif (signature protein motif), potential open reading frame (ORFs) can be detected.

The cDNA sequence that obtains then is designed primer to carrying out sxemiquantitative PCR to confirm the tissue expression pattern.Selecteed product can be used to separate full length cDNA clone so that further analyze.SSCP-PCR (the strand formation polymorphism-PCR) amplification of primer to being used to genomic dna.For example, determine the connection of each PCR product and the mark that has been connected by one group of inner special mouse of backcrossing.This can produce the collection of illustrative plates of new gene and can be used as the mutational site of the mouse of identifying existing collection of illustrative plates and the resource of homologous people's disease gene.SSCP-PCR synthetic oligonucleotide primer, by a little fragment of pcr amplification (100-200bp) (M.Orita etc., " form the detection of multifarious people's dna polymorphism as strand by gel electrophoresis ", Proc.Natl.Acad.Sci.USA 86:2766-2770 (1989); (M.Orita etc., " carrying out detection fast and delicately ", Genomics5:874-879 (1989)) in the dna polymorphism point mutation with the polymerase chain reaction.

The cDNA fragment that is sheared can be carried out radio-labeling with this field technique known, confirms that mRNA distributes and understands size and the advantage that corresponds to full length mRNA so that carry out careful (northern) trace of promise or in situ hybridization (in situ) with probe.Probe can be used to screen a cDNA library and carry out determining of more reliable and more complete sequence with separating clone.The probe that is labeled also can be used to any other purpose, for example studies in external expression.

III. special group and degenerated primer mixture

Another aspect of the present invention is primer and the degenerated primer mixture that is fit to the panel of practical application of the present invention, and these comprise:

(1) containing the primer of a panel of 16 primers of sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO:16), is among 4 deoxyribonucleotide A, C, G or the T one at this N;

(2) contain the primer of a panel of 64 primers of sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ IDNO:17);

(3) contain the primer of a panel of 256 primers of sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ IDNO:18);

(4) contain the primer of a panel of 1024 primers of sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQID NO:19);

(5) contain the primer of a panel of 4096 primers of sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO:20);

(6) contain the primer of a panel of 3 primers of sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:3);

(7) containing the primer of a panel of 12 primers of sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO:4), is 1 deoxyribonucleotide selecting from the group that A, C and G form at this V;

(8) contain the primer of a panel of 48 primers of sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:5);

(9) contain the primer of a panel of 3 primers of sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:6);

(10) contain the primer of a panel of 12 primers of sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO:7);

(11) contain the primer of a panel of 48 primers of sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ IDNO:8);

(12) contain 4 different oligonucleotides, each has the primer of the panel of sequence G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:22);

(13) contain 16 different oligonucleotides, each has the primer of the panel of sequence G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO:23);

(14) contain 64 different oligonucleotides, each has the primer of the panel of sequence T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ ID NO:24);

(15) contain 256 different oligonucleotides, each has the primer of the panel of sequence C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO:25);

(16) contain 1024 different oligonucleotides, each has the primer of the panel of sequence G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N (SEQ ID NO:26);

(17) contain 4096 different oligonucleotides, each has the primer of the panel of sequence A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO:27);

(18) comprise and have sequence A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G--C-A-G-G-A-A-T-T-a degenerated primer mixture of the mixture of 3 primers of T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:2), each in 3 primers with equimolar measure existing;

(19) comprise and have sequence A-a degenerated primer mixture of the mixture of 12 primers of A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ IDNO:4), each in 12 primers with equimolar measure existing;

(20) comprise and have sequence A-a degenerated primer mixture of the mixture of 48 primers of A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:5), each in 48 primers with equimolar measure existing;

(21) comprise a degenerated primer mixture of the mixture of 3 primers with sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ IDNO:6), each in 3 primers with equimolar measure existing;

(22) comprise a degenerated primer mixture of the mixture of 12 primers with sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQID NO:7), each in 12 primers with equimolar measure existing;

(23) comprise the have sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N one degenerated primer mixture of 48 primer mixtures of (SEQ ID NO:8), each in 48 primers with equimolar measure existing.

IV. certain embodiments of superiority particular embodiment

Embodiment 1: the application of improving one's methods

On the basis of the observation of the eukaryote mRNA s that is based on nearly all poly of containing (A) tail of improving one's methods of the present invention, but be different from difference and show (Liang, P and A.B.Pardee (1992), difference by polymerase chain reaction method eukaryote messenger RNA(mRNA) shows, Science 257:967-971), method of the present invention is only fixed a site in conjunction with the specificity of tail with primer on each mRNA, needn't be divided into Ya Ku to mRNAs again.Improved method has been proved to be in three embodiment of Fig. 1, Fig. 2 and Fig. 8.

Summarize and say, double-stranded cDNA produces from enrich poly (A) tenuigenin mRNA, and mRNA is the equimolar mixture of anchor primer with 48 all 5 '-vitamin Hs of the initial reverse transcription of a cover, (Gubler from the sample extraction of purpose tissue, U. and the simple but method in very effective generation cDNA library of (1983) one of B.Hoffman, Gene 25:263-269) (Schibler, K., M.Tosi, A.C.Pittet, the tissue-specific expression of the amylase gene of L.Fabiani and P.K.Wellauer (1980) mouse, J.Mol.Biol.142:93-116).A cover primer that is fit to like this is A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:5), here V is A, C or G, and N is A, C, G or T.A member of these 48 anchor primer mixtures initial in sample synthetic in a 3 ' terminal fixed position of each all copy of mRNA species, therefore, define 3 ' terminal point of each species, produced the double-stranded cDNA of vitamin H.

With cutting the double-stranded cDNA sample of each vitamin H (acyl group) by recognition sequence CCGG restriction enzyme MspI.Then on the substrate of antibiosis protein chain mycin bag quilt, by the 3 ' fragment of separating cDNA of catching of vitamin H (acyl group) cDNA.Suitable bag is comprised the polymeric beads of microtiter plate, PCR pipe, polystyrene bead, paramagnetic and the sintered glass particle of paramagnetic by the substrate of antibiosis protein chain mycin.Preferred bag by antibiosis protein chain mycin substrate be the paramagnetic polymeric beads suspension (Dynal, Inc, Lake Success, NY).

The washing bag is by the substrate of antibiosis protein chain mycin with after catching the cDNA fragment of vitamin H (acyl group); discharge cDNA fragment product by NotI digestion; the 8-nucleotide sequence place cutting of NotI in anchor primer, but seldom partly cut at the mRNA that derives from of cDNAs.3 ' MspI-NotI fragment has consistent length for each mRNA species, directly to be connected the plasmid pBC SK that enters with ClaI-, NotI-cutting with respect to the anti-significance direction of carrier T3 promotor ⁺(CA), and product is used to transformed into escherichia coli (E.coli) SURE cell (Stratagene) for Stratagene, LaJolla.Ligation regeneration NotI site, but do not have the MspI site.Contain and surpass 5 * 10 ⁵Recombinant chou, the 3 ' end of guaranteeing all mRNAs have 0.001% or each library of offers additional possibilities, are repeatedly described.The preparation of carrying out plasmid from the cDNA library of each sample just under study for action.

Digest equal portions in each library with MspI, MspI is by several sites cutting in parent vector and stay 3 ' complete cDNA insertion sequence simultaneously and its flanking sequence, comprises the T3 promotor, realizes linearizing.(MEGAscript Kit, Ambion) incubation produces and to contain known carrier sequence, transcribes but the clone who has deleted the MspI that derives from original cDNA s and NotI site inserts segmental anti-meaning cRNA for product and T3 RNA polymerase.

This step is avoided deriving from not having and inserts the pollution in various degree that plasmid is transcribed, because the difference of primer is competed, does not have and inserts the variation that plasmid will cause the efficient of the later PCRs of different samples.Yet, a MspI site is arranged between the multi-link zone C laI of parent vector and NotI site, therefore, the MspI digestion step has been got rid of to derive from does not have the 5 ' label of transcribing cRNAs that inserts plasmid, makes them insert in following product amplification step.By with the RNase incubation of no DNase, plasmid DNA is transcribed the mixture from anti-meaning cRNA and is removed.

In this stage, the preparation of each cRNA is carried out in one three step mode.In the first step, convert the cRNA of 250ng to first chain cDNA with 5 ' RT primer (5PPIMER in Fig. 1,2 and 8) A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO:14).CDNA product at the second step 400pg is used as pcr template, with each and general 3 ' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:47) pairing in 45 ' the PCR primers of G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:22) form, in four isolating reactions, use following program:

94 ℃, 15 seconds;

65 ℃, 15 seconds;

72 ℃, 60 seconds

20 circulations.

In the 3rd step, the product in each inferior storehouse further is divided into 64 Ya Yaku (2ng among the 20 μ l), with 100ng fluorescently-labeled " general " 3 ' PCR primer, nucleic acid oligomer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:47) is connected to 6-FAM by conjugation and suitable form is on 5 '-PCR primer of C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO:25), carries out the PCR reaction second time.Use following program:

94 ℃, 15 seconds;

X ℃, 15 seconds;

72 ℃, 30 seconds

30 circulations.

This comprises annealing steps, and the Tm of a little higher than each 5 ' the PCR primer of its temperature X makes the people minimize for the mispairing primer, improves the height fidelity simultaneously and duplicates.Each polymerase chain reaction step is carried out in the presence of TaqStart antibody (Clonetech).

For the product that derives from last polymerase chain reaction step in each tissue sample, on a series of denatured DNA sequencing gels, analyze with automatic ABI Prizm 377 sequenators.Collect data and standardized amplitude and migration with genescan software package (ABI).For N ₁In 4 inferior storehouses that 5 ' PCR primer is set up each is carried out this a series of complete reactions and is produced the inferior storehouse of 64 products, carries out these a series of complete reactions for N45 ' PCR primer sets completely and produces 256 inferior storehouses of product altogether.

Summarize and say, in the embodiment that this is improved one's methods (Fig. 2), non-brief primer 5 ' RT primer with 5PRIMER (SEQ ID NO:14) form, utilize ThermoScript II from cRNA, to produce the inferior storehouse of a cDNA, as 5 '-PCR primer and 3 '-PCR primer (SEQ ID NO:47), utilize Taq archaeal dna polymerase (20 circulations) in PCR to produce the inferior storehouse of double-stranded cDNA with 5 ' PCR primer 5PRIMERN1 (SEQ ID NO:11).Last PCR 2ng dna profiling and each 5PRIMER ₃N ₁N ₂N ₃N ₄3 ' each 100ng of PCR primer that primer (SEQ ID NO:25) and conjugation are linked on the 6-FAM carries out 30 circulations.

Analysis derives from hungry serum (Serum-starved) (Fig. 3, the A of panel) and adds 2 mRNA samples of serum (Fig. 3, the B of panel) people's the sick cell of MG63 osteosarcoma.With 5 '-PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-G-G-T-G, SEQ ID NO:42) and at 5 '-end mark the 6-Fluoresceincarboxylic acid is arranged (6FAM, the pairing of ABI) " general " 3 ' primer (SEQID NO:47) has produced data presented.The analyzed separation of gel electrophoresis PCR product on 4.5% acrylamide gel, and on the ABI377 automatic sequencer, obtain fluorescence data.With genescan software package (Perkin-Elmer) analytical data.In 3 shown in the above panels, the relative abundance of mark PCR product is plotted figure (Y-axle=relative fluorescence primitive unit cell) to product base length.The height repeatability of present method is displayed in the following panel, and it shows with what the genescan software package carried out relative expression's level between sample and relatively derives from overlapping panel (A) and data (B).

Main application of the present invention is that more two or more tissue sample mRNA express profile.We have compared the influence for the panel's serum starvation that produces product/interpolation experiment.Right product great majority have comparable amplitude to derive from common mobile example.Product is less than 10% amplitude that has by two or more factor difference, and these almost are being homoeomerous between species that check by adding that serum inductive species and those are added.

Many products, some are differently represented in two panels in them, move and match based on the DSTs of the prediction of extracting from GenBank locational, have so just produced a candidate's identity.In order to detect these candidates' identity, synthetic and 5PRIMER ₃N ₁N ₂N ₃N ₄The oligonucleotide that (SEQ IDNO:25) is corresponding, 14 additional Nucleotide using the sequence that derives from adjacent end MspI site in the GenBank sequence are 3 ' the terminal extension.Use N ₁CDNA makes substrate, and the 3PRIMER (SEQ ID NO:47) of these and fluorescence matches in PCRs.

PCR step of embodiment 2 usefulness or two PCR steps: PCR product analysis, brief specificity in present method particular embodiment

The advantage of particular embodiment that comprises the basic skills of two PCR steps utilizes the cleer and peaceful interpolation serum of hungry blood MG63 cell to be proved to be.(primer (SEQ ID NO:14) with a non-brief form is 5PRIMER utilizes ThermoScript II to produce a cDNA storehouse from cRNA for Fig. 1, the variation of two PCR steps Fig. 2) for basic skills; As 5 '-primer and 3 ' primer (SEQ ID NO:47), in PCR, utilize the Taq archaeal dna polymerase to produce the inferior storehouse of double-stranded cDNA with 5PRIMER1 (SEQ ID NO:22).In the modification of first PCR step, be that in 4 N1 primers of 5PRIMERN1 (SEQ ID NO:22) one transcribes by initial with form, utilize ThermoScript II to produce the inferior storehouse of 4 cDNA from cRNA.In two methods, last PCR 2ngDNA template, 5, each 100ng of 3 ' PCR primer (SEQ ID NO:47) of PCR primer (SEQ ID NO:25) and mark 6-FAM carries out 30 circulations.

The PCR fragment of mark goes up at automated DNA sequenator (ABI377) and analyzes with the genescan software package by electrophoresis method.The result as shown in Figure 4.Derive from primer 109T (C-G-A-C-G-G-T-A-T-C-G-G-T-G-C-A, SEO ID NO:43) and 45A (C-G-A-C-G-G-T-A-T-C-G-G-A-G-C-A, SEO ID NO:44) data are proved by the sample (Fig. 4 B, 4D, 4F and 4H) of hungry serum (Fig. 4 A, 4C, 4E and 4G) and interpolation serum only in N1 position (runic) difference.

The PCR product with 45A produces with 109T seems that the template that the modification of almost same PCR step produces is identical (comparison diagram 4A and Fig. 4 C, Fig. 4 B and Fig. 4 D).Contrast, the product that derives from the follow-up PCR detection of the template that produces with two PCR step method is diverse (comparison diagram 4E and Fig. 4 G, Fig. 4 F and Fig. 4 H), therefore, two PCR steps that present method embodies provide substantial improvements for the method that is suitable in the past.

Embodiment 3: with a PCR step or two PCR steps brief specificity in present method particular embodiment: clone and sequencing data.

Method of the present invention is used or being embodied on the cleer and peaceful processing serum of the hungry blood MG63 cell of a PCR step (Table I) or two PCR steps (Table II) carried out.In the shown experiment of Table I, with 4 N1 5 ' PCR primers of that cover (SEQ ID NO:22), transcribe by initial, utilize ThermoScript II to produce to derive from the inferior storehouse of 4 cDNA of cRNA.For Table II, derive from a cDNA in the inferior storehouse of a cRNA with the ThermoScript II generation with 5 ' the RT primer (SEQ ID NO:14) of a non-parsimony.With 5 ' PCR primer (SEQ ID NO:22) and 3 ' PCR primer (SEQ ID NO:47), utilize the Taq archaeal dna polymerase in PCR (20 circulations), to produce the inferior storehouse of double-stranded cDNA.Last PCR in Table I and Table II, with 2ng input cDNA template, whole 256 5 '-3 '-PCR primer (SEQ ID NO:47) of PCR (SEQ ID NO:25) primer series and 6FAM mark matches, is carried out equally.From the PCR reaction and display, the molecule that differently is conditioned to be identified with the purpose that checks order and is separated in order to clone.

In data search subsequently, determine clone and gene identification, obtain dna sequence data with the BLAST algorithm.In table,, listed and found and the accurate clone who mates of known Human genome by gene name and GenBank location identity.By being evaluated at the fidelity of using the brief step of 5PRIMERN1 (SEQ ID NO:22) in reverse transcription (Table I) or the PCR reaction (Table II) making table with respect to the sequence of GenBank sequence clone coupling in the N1 position.In two-stage process, 5/22 is cloned in N1 position (being actually at random) goes up correct coupling, and in three step programs, all clones are found with the data of corresponding GenBank sequence and correctly mate.

Table I: the brief specificity (P65) of a PCR step

Gene name GenBank location identity N1 location matches

Nma

CDE1 is conjugated protein

The laminin receptor homologue

The C albumen that U1 snRNP-is special

Ubiquitin

MAD-3

Alpha-tubulin

Id1

NNMT

BFGF

SC35

Ribosomal protein S14

Ribosomal protein L 30

Na/K?ATPase?B3

Ribosomal protein L 37A

IRF-2

SRp20

GLO-I I

The pim-1 oncogene

Endothelin-1

Metallothionein(MT) II

The CRP3 homologue

Table II: the brief specificity (P66) of two PCR steps

Gene name GenBank location identity N1 location matches

MAD-3

Id1

Na/K?ATPase?B3

The pim-1 oncogene

Endothelin-1

Ribosomal protein S20

Ribosome protein S 10

GADD45

AP-2

Beta-2-microglobulin

RDC-1

The automatic antigen of 56K

NFKB1

Lon proteolytic enzyme sample albumen

Nucleotide binding protein

The nesidioblastoma gene

Histone 2A.2

5 gene products that attention highlights with black matrix, MAD-3, Idl, Na/KATPase B3, Pim-1 oncogene and endothelin-1 are separated in two real dangers, and each situation of two PCR step method is at N ₁The position has produced a coupling, and a PCR step method is not mated like this.Therefore two PCR steps provide a substantial improvements for the method that is suitable in the past.

The improvement result that embodiment 4 usefulness, one vitamin Hs (acylations) anchor primer obtains

As mentioned above, in a preferred embodiment, anchor primer is changed by vitamin H (acyl group) at 5 ' terminal (comparison diagram 1 and Fig. 2).The cDNA fragment of vitamin H (acylations) can preferably be wrapped by the paramagnetic beads of antibiosis protein chain mycin (Dynal) with a bag by antibiosis protein chain mycin substrate capture.Fig. 5 has compared result who obtains with standard method and the method that obtains with magnetic bead anchor primer mark.

Alternate embodiments (see figure 2) with standard techniques (as shown in Figure 1) and magnetic bead, extraction derives from the mouse that haloperidol was handled in a series of timed interval (0,0.75,7 hour, 10 and 14 days), the 2gmRNA aliquot of striatal 5 sample separation, the construction cDNA library.The result is shown in Fig. 5 A-E (standard) and 5F-J (magnetic bead).Deriving from the result that two kinds of situations of 3 ' the PCR primer (SEQ ID NO:47) of 5 ' PCR primer 170G (C-G-A-C-G-G-T-A-T-C-G-G-G-G-T, SEQ ID NO:45) and mark 6-FAM compare is shown.The relative abundance of mark PCR product is to the length (base pair) of PCR product drawn (Y-axis be flat fluorescent) arbitrarily.The data that derive from magnetic bead library (Fig. 5 F-J) compare with the data that derive from standard library (Fig. 5 A-E), under a series of timed interval (consistence that segmental similarity and strength values are all arranged) and at sample room, has weak point (100-125) fragment of bigger replicability and few tangible vacation.

Embodiment 5 is in the proof of three one step process neutral lines: the dependency of the cDNA concentration of the peak height of PCR product and input

In order to determine the scope of linear amplification, single cRNA species are divided into 4 independently inferior storehouses of cRNA, and handle with method as shown in Figure 2 of the present invention.The result as shown in Figure 6.The peak height of corresponding synthetic RNA (with relative flat fluorescent) is measured, and simultaneously the concentration of 4 samples of input is drawn.Data presented is 3 multiple mean values of each sample.The mistake thick stick represents ± mistake of a standard means.

A SalI-NotI cDNA fragment (SEQ ID NO:51) is cloned into carrier pBC SK ⁺, by produce the peak that cRNA and linearizing, synthetic cRNA are fabricated a known length among PCR of generation (492bp) from the T3 promoter transcription.Use N ₀Primer (SEQ ID NO:14) is before reverse transcription, and the amount (0,25,100 or 250pg) that changes cRNA is introduced in the cDNA storehouse of a 250ng.As template, use 5 ' PCR primer (SEQ ID NO:22) and 3 ' PCR primer (SEQ ID NO:47) to carry out the PCR reaction with the cDNA of 400pg respectively.In a last PCR reaction, use the cDNA of 2ng equal portions with 5 ' PCR primer 2 21C (C-G-A-C-G-G-T-A-T-C-G-G-C-T-C-A, SEQ ID NO:46) and 3 ' PCR primer (SEQ ID NO:47).Proved for a given types of organization peak height of PCR product and be proportional with the RNA concentration of importing in the described result of Fig. 6.

Aforementionedly be intended to graphic illustration the present invention, but not limited.Under the situation that discord real spirit and scope of the present invention deviate from, a large amount of variation of the present invention and modification may be effective.

Sequence table＜110〉Hasel, Karl W.

Hilbush，Brian?S.<120>Method?For?Indexing?And?Determining

The?Relative?Concentration?of?Expressed?Messenger?RNA<130>98，429-A<140><141>1999-10-14<150>US09/186,869<151>1998-11-04<160>51<170>PatentIn?Vet.?2.0<210>1<211>14<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>1aactggaaga?attc 14<210>2<211>14<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>2gaattcaact?ggaa 14<210>3<211>46<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>3aactggaaga?attcgcggcc?gcaggaattt?tttttttttt?tttttv 46<210>4<211>47<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>4aactggaaga?attcgcggcc?gcaggaattt?tttttttttt?tttttvn 47<210>5<211>48<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>5aactggaaga?attcgcggcc?gcaggaattt?tttttttttt?tttttvnn 48<210>6<211>47<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>6gaattcaact?ggaagcggcc?cgcaggaatt?tttttttttt?ttttttv 47<210>7<211>48<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>7gaattcaact?ggaagcggcc?cgcaggaatt?tttttttttt?ttttttvn 48<210>8<211>49<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>8gaattcaact?ggaagcggcc?cgcaggaatt?tttttttttt?ttttttvnn 49<210>9<211>116<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：multiple?cloning?site<400>9gagctccacc?gcggtgtcac?gactatctgc?ggccgcatgc?ccgggaatgg?cgcctcgaga 60cgtctttatc?gataccgtcg?acctcgaact?cgagacgtcc?cgggcgccta?ggtacc 116<210>10<211>113<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：multiple?cloning?site<400>10gagctcgttt?tcccagtcac?gactatctgc?ggccgcatgc?ccgggaatgg?cgcctcgaga 60cgttatcgat?tagcctgact?gaagactcga?gacgtcccgg?gcgcctaggt?acc 113<210>11<211>113<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：multiple?cloning?site<400>11gagctcgttt?tcccagtcac?gactatctgc?ggccgcatgc?ccgggaatgg?cgcctcgaga 60cgtctatatc?gattagcctg?actgaagact?cgagacgtcc?cgggctaggt?acc 113<210>12<211>62<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：multiple?cloning?site<400>12gcggccgcat?agatctgata?tcggatcctc?accacagagc?tcagtgagag?agatctctcg 60ag 62<210>13<211>62<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：multiple?cloning?site<400>13gcggccgcat?ccatgggata?tcgcatgctc?accacagtcg?acagtgagag?ccatggctcg 60ag 62<210>14<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>14aggtcgacgg?tatcgg 16<210>15<211>17<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>15aggtcgacgg?tatcggn 17<210>16<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>16aggtcgacgg?tatcggnn 18<210>17<211>19<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>17aggtcgacgg?tatcggnnn 19<210>18<211>20<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>18aggtcgacgg?tatcggnnnn 20<210>19<211>21<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>19aggtcgacgg?tatcggnnnn?n 21<210>20<211>22<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>20aggtcgacgg?tatcggnnnn?nn 22<210>21<211>15<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>21ggtcgacggt?atcgg 15<210>22<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>22ggtcgacggt?atcggn 16<210>23<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>23gtcgacggta?tcggnn 16<210>24<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>24tcgacggtat?cggnnn 16<210>25<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>25cgacggtatc?ggnnnn 16<210>26<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>26gacggtatcg?gnnnnn 16<210>27<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>27acggtatcgg?nnnnnn 16<210>28<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>28agctctgtgg?tgaggatc 18<210>29<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>29gctctgtggt?gaggatcn 18<210>30<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>30ctctgtggtg?aggatcnn 18<210>31<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>31tctgtggtga?ggatcnnn 18<210>32<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>32ctgtggtgag?gatcnnnn 18<210>33<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>33tgtggtgagg?atcnnnnn 18<210>34<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>34gtggtgagga?tcnnnnnn 18<210>35<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>35tcgactgtgg?tgagcatg 18<210>36<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>36cgactgtggt?gagcatgn 18<210>37<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>37gactgtggtg?agcatgnn 18<210>38<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>38actgtggtga?gcatgnnn 18<210>39<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>39ctgtggtgag?catgnnnn 18<210>40<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>40tgtggtgagc?atgnnnnn 18<210>41<211>18<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>41gtggtgagca?tgnnnnnn 18<210>42<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>42cgacggtatc?ggggtg 16<210>43<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>43cgacggtatc?ggtgca 16<210>44<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>44cgacggtatc?ggagca 16<210>45<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>45cgacggtatc?gggggt 16<210>46<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>46cgacggtatc?ggctca 16<210>47<211>15<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>47gagctccacc?gcggt 15<210>48<211>16<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>48gagctcgttt?tcccag 16<210>49<211>22<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>49gtcttcagtc?aggctaatcg gn 22<210>50<211>22<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>50cctcgaggtc?gacggtatcg?gn 22<210>51<211>481<212>DNA<213>Artificial?Sequence<220><223>Description?of?Artificial?Sequence：synthetic

primer<400>51gtcgacggta?tcggctcaag?tgactgactg?tctagaactt?taccattacg?gagagatgat 60gatcagtaac?caagattatc?ttggactatc?tttaggttct?ttaaaaaaac?tgcttattac?120caacctttgt?agctgaccta?agatctttgt?gcctgttatg?taaaaagttt?ggaatgtatt?180gttaaactta?gccaacgact?ggcttttcag?cagtgctcaa?aagaagagta?tcatcagctg?240gagattttcc?tgctatgctg?tagcctacct?ccccgatgtc?ctttccgcta?tatttggcaa?300atgtattgat?ttatggtctt?ttgttctatg?gctataagac?tgcgtgtaaa?cctctttcac?360agtagaacat?gtaattctgg?gaaacccgaa?tctctgttac?taagcactat?tcactcaaag?420ttgcctcaga?ataaactttc?tttgggtttt?aaaaaaaaaa?aaaaaaaatt?cctgcggccg?480c 481

Claims

1. improving one's methods of the special evaluation of a synchronizing sequence that carries out mRNAs in a mRNA storehouse, it comprises the steps:

(a) prepare a double-stranded cDNA storehouse with a blended anchor primer from a mRNA storehouse, each anchor primer contains one 5 ' terminal and one 3 ' end, and it comprises one section sequence of (i) 7-40T residue; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 ' terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer be positioned toward with by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer, it inserts 6 above bases of identification by between the sequence of first restriction enzyme cleavage site and T residue; (v) from by-V ,-V-N and-each anchor primer phase transformation residue of selecting the group that V-N-N forms is positioned in 3 '-end, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N;

(b) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of this second restriction enzyme identification has formed a double-stranded cDNA library of molecules that first and second ends are arranged respectively;

(c) each the double-stranded cDNA molecule that derives from step (b) is inserted into into carrier in a direction, it is anti-meaning for the special promotor of phage, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', 3 ' flanking vector of 3 ' end of 5 ' the terminal and sense strand that inserts the cDNA sense strand, and the described structure that one 3 ' flanking vector sequence arranged has 15 length of nucleotides in described first restriction endonuclease sites and qualification at least between transcription initiation site in described promotor;

(d) insert the carrier transformed host cells with the cDNA that is cut, contain the carrier that the clone inserts with generation;

(e) with at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the special promoter sequence of phage, but the structure storehouse that the digestion with restriction enzyme of sequence produces in step (c) on the energy identification carrier, so just produced the linear segment of the cDNA molecule that contains insertion, the linear fragment of Chan Shenging has one at least in 5 ' the flanking vector sequence of carrier 5 ' to 15 Nucleotide of second end of double-stranded cDNA molecule like this;

(g) with a ThermoScript II and 15-30 Nucleotide long and comprise by with 5 ' reverse transcription primer of 5 ' flank body sequence complementary nucleotide sequence, produce the first chain cDNA by transcribing cRNA;

(h) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA, the polymerase chain reaction is that 15-30 Nucleotide is long with first 3 ' PCR primer for the first time, it and cutting the enzyme site and determining 3 ' flanking vector sequence complementation between the transcription initiation site by the special promotor of phage in first is restricted, first 5 ' PCR primer has been defined-N ₁3 '-end of forming, wherein " N " is among four kinds of deoxyribonucleotide A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier preface complementation of 5 ' flank, simultaneously, the complementation of first 5 ' PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, in different primers of this first 5 ' PCR primer are used in four inferior storehouses of difference each;

(i) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product, second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, it and determine 3 ' flanking vector sequence complementation between the transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (h), " x " is from 1 to 5 integer, this primer length is that 15-30 Nucleotide is long, and and 5 ' flanking vector sequence complementation, simultaneously complementary the extension with Nucleotide quantity of primer is equivalent to the special insertion Nucleotide that cRNA is inserted in " x "+1, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and each inferior storehouse has 4 in second series in the inferior storehouse of first cover ^xYa Ku;

2, according to the process of claim 1 wherein that a biotin moiety linked on the anchor primer by conjugation.

3, according to the method for claim 2, wherein said biotin moiety is linked anchor primer 5 ' end by conjugation.

4, according to the method for claim 2, wherein said first restrictive cDNA is from by isolating by the remainder that the proteic substrate of antibiosis contacts cDNA among first restrictive cDNA step b claim 1 with a bag.

5, describedly connect by thiophosphatephosphorothioate according to the process of claim 1 wherein at 3 ' of first 5 ' PCR primer 3 terminal Nucleotide.

6, describedly connect by thiophosphatephosphorothioate according to the process of claim 1 wherein at 3 ' of second 5 ' PCR primer 3 terminal Nucleotide.

7, describedly be connected by thiophosphatephosphorothioate according to the process of claim 1 wherein at 3 ' of first and second 5 ' PCR primer 3 terminal Nucleotide.

8, according to the process of claim 1 wherein that the primer of described second PCR reaction is connected a fluorescence labels by conjugation.

9, method according to Claim 8, wherein said fluorescence labels are to select from the composition of following group:

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 6-carboxylic acid, 3 ', 6 '-dihydroxyl-6-Fluoresceincarboxylic acid;

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 5-carboxylic acid, 3 ', 6 '-dihydroxyl-5-Fluoresceincarboxylic acid;

Spiral (isobenzofuran-1 (3H), 9 '-(9H)-xanthene)-3-one, 3 ', 6 '-dihydroxyl fluorescein;

9-(2,5-dicarboxyl phenyl)-3,6-two (diformazan aminophenyl)-xanthene-6-carboxyl tetramethylrhodamin;

3,6-diamino-9-(2-carboxyl benzene is base just)-xanthene;

Spiral [isobenzofuran-1 (3H), 9 '-xanthene]-6-carboxylic acid, 5 '-two chloro-3 ', 6 '-dihydroxyl-2 ', 7 '-dimethoxy-3-oxo-;

1H, 5H, 11H, 15H-xanthene o[2,3,4-ij:5,6,7-i ' j '] two quinolizines-8-,-(2,4-two sulfur phenenyls)-2,3,6,7,12,13,16,17-octahydro, inner salt;

6-((4-4-two fluoro-5,7-dimethyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino) caproic acid;

6-((4,4-two fluoro-1,3-dimethyl-5-(4-p-methoxy-phenyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionyl) amino)-caproic acid;

6-(((4-(4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) benzene oxygen) acetyl) amino)-caproic acid;

4,4-two fluoro-4-boron-3a, 4a-phenodiazine-s-indacene-3-valeric acid;

4,4-two fluoro-5,7-dimethyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid;

4,4-two fluoro-5-phenyl-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid;

4,4-two fluoro-5 (4-phenyl-1,3-butadiene base)-4-boron-3a, 4a-phenodiazine-s-indacene-3-propionic acid;

6-(((4,4-two fluoro-5-(2-thienyl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) styryl oxygen) ethanoyl) hexosamine;

6-(((4,4-two fluoro-5-(2-pyrryl)-4-boron-3a, 4a-phenodiazine-s-indacene-3-yl) styryl oxygen) ethanoyl) hexosamine;

9-(2,4 (or 2,5)-dicarboxyl phenyl)-3,6-two (dimethylamino)-xanthene, inner salt; And

4,7,2 ', 4 ', 5 ', 7 ' chlordene-6-Fluoresceincarboxylic acid and 4,7,2 ', 7 ' tetrachloro-6-Fluoresceincarboxylic acid.

10, according to the process of claim 1 wherein that described host cell is a Bacillus coli cells.

11, according to the process of claim 1 wherein that described phase transformation residue in step (a) is-V-N-N.

12, according to the process of claim 1 wherein that described phase transformation residue in step (a) is-V-N.

13, according to the process of claim 1 wherein that described phase transformation residue in step (a) is-V.

14, according to the process of claim 1 wherein that described " x " in step (i) is 3.

15, according to the process of claim 1 wherein that described " x " in step (i) is 1.

16, basis the process of claim 1 wherein that described phase transformation residue in step (a) is-V-N-N, and " x " in step (i) is 3.

17, basis the process of claim 1 wherein that described phase transformation residue in step (a) is-V, and " x " in step (i) is 2.

18, according to the process of claim 1 wherein that described each anchor primer has 18 T residues in T residue district.

19, according to the process of claim 1 wherein that first stuffer length of described anchor primer is 14 residues.

20, according to the process of claim 1 wherein that the sequence of described first stuffer is G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO:2).

21, according to the process of claim 1 wherein that the special promotor of described phage screens from being started the molecular group by T3 promotor, T7 promotor and SP6.

22, according to the process of claim 1 wherein that the special promotor of described phage is the T3 promotor.

23, according to the process of claim 1 wherein that the primer sequence of transcribing design of described cDNA from cRNA is A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO:14).

24, according to the process of claim 1 wherein that described carrier is the plasmid pBC SK with ClaI and NotI cutting ⁺, 3 ' PCR primer is G-A-G-C-T-C-C-A-C-C-G-C-G-T (SEQ ID NO:47) in step (h) and (i).

25, according to the process of claim 1 wherein that described carrier is the plasmid grain pBC SK with ClaI and NotI cutting ⁺, 3 ' PCR primer is G-A-G-C-T-C-G-T-T-T-T-C-C-C-A-G (SEQ ID NO:48) in step (h) and (i).

26, according to the process of claim 1 wherein that second restriction enzyme of a 4-nucleotide sequence of described identification is MspI.

27, according to the process of claim 1 wherein that second restriction enzyme of a 4-nucleotide sequence of described identification is from by screening the group that MboI, DpnII, Sau3AI, Tsp509I, HpaII, BfaI, Csp6I, MseI, HhaI, NlaIII, TaqI, MspI, MaeII, Sau3AI, Bg1II and HinP1I formed.

28, according to the process of claim 1 wherein that first restriction enzymes of 6 above bases of described identification is from by screening the group that AscI, BaeI, FseI, NotI, PacI, PmeI, PpuMI, RsrII, SapI, SexAI, SfiI, SgfI, SgrAI, SrfI, Sse8387I and SwaI formed.

29, according to the process of claim 1 wherein that first restriction enzyme of 6 above bases of described identification is NotI.

30, according to the process of claim 1 wherein that the described restriction enzyme that is used in the step (e) has an identification that contains a nucleotide sequence of the 4-nucleotide sequence that is used in second restriction enzyme in the step (b).

31, according to the method for claim 30, wherein said second restriction enzyme is MspI, and the restriction enzyme that is used in the step (e) is SmaI.

32, according to the method for claim 30, wherein said second restriction enzyme is TaqI, and the restriction enzyme that is used in the step (e) is XhoI.

33, according to the method for claim 30, wherein said second restriction enzyme is HinP1I, and the restriction enzyme that is used in the step (e) is NarI.

34, according to the method for claim 30, wherein said second restriction enzyme is MaeII, and the restriction enzyme that is used in the step (e) is AatII.

35, according to the method for claim 30, wherein said second restriction enzyme is Sau3AI, and the restriction enzyme that is used in the step (e) is Bg1II.

36, according to the method for claim 30, wherein said second restriction enzyme is NlaIII, and the restriction enzyme that is used in the step (e) is NcoI.

37, be appropriate to the carrier that the described method of claim 1 is used, wherein the carrier of step (c) is a circular DNA molecule form, it has the first and second carrier restriction endonuclease sites at carrier padding sequence flank, and further is included in the step of the first and second carrier restriction endonuclease sites with the digested vector of restriction enzyme cut vector.

38, according to the carrier of claim 37, the interior carrier that wherein said padding sequence includes between the first and second carrier restriction endonuclease sites is filled restriction endonuclease sites.

39, according to the carrier of claim 38, wherein said step (e) comprises the vector digestion of filling the restriction endonuclease sites cut vector with a restriction enzyme in interior carrier.

40, a kind of carrier, it is from by plasmid pBC SK ⁺/ DGT1, pBS SK ⁺/ DGT2, pBSSK ⁺/ DGT3, pBC SK ⁺/ DGT4 and pBS SK ⁺Screen in the group that/DGT5 forms

41, a kind of carrier, it contains screening of a multiple clone site from the group that is made up of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

42, described for poly adenosine mRNA species according to the process of claim 1 wherein, the mRNA storehouse is by abundant.

43, according to the process of claim 1 wherein that described amplified fragments is undertaken by the electrophoresis showed product at the compartment analysis of step (j).

44, according to the method for claim 43, wherein said intensity at the product that shows behind electrophoresis abundance of the corresponding mRNAs of product almost and in original stock is ratio.

45, according to the method for claim 43, after it further was included in electrophoresis, the intensity that corresponds to mRNA from product was determined the step of each mRNA relative abundance original stock.

46, according to the method for claim 43, wherein said by the segmental step of electrophoretic separation analysis polymerase chain reaction (PCR) amplification product, be included in the segmental electrophoresis at least two gels.

47, according to the method for claim 40, it further may further comprise the steps:

(k) wash-out at least one with derive from the corresponding cDNA of mRNAs in the electrophoretogram, wherein the representative band that appears at the mRNAs of 3 ' in the sample-end is shown;

(l) cDNA of amplification wash-out in a polymerase chain reaction;

(m) cDNA of clonal expansion enters in the plasmid;

(n) production derives from the same clone's of plasmid the corresponding DNA of DNA;

(o) order-checking clone cDNA.

48, the special mRNAs of synchronizing sequence identifies improves one's methods for carrying out in a mRNA storehouse of may further comprise the steps:

(a) separate a mRNA storehouse;

(b) prepare a double-stranded cDNA storehouse with a blended anchor primer from the mRNA storehouse, wherein each anchor primer has one 5 ' terminal and one 3 ' end, and comprises (i) sequence from 7 to 40T residues; (ii) be identified the site of first restriction enzyme cutting of 8 bases, this cleavage site is relevant with the sequence of T residue towards 5 '-terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer be positioned toward with by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer inserts 6 above bases of identification by between the sequence of first restriction enzyme cleavage site and T residue; (v) by the 3 ' terminal-V that is positioned in each anchor primer ,-V-N or-phase transformation residue that one of V-N-N limits, wherein V is the deoxyribonucleotide that screens from the group that is made up of A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N;

(c) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of second restriction enzyme identification has formed a double-stranded cDNA library of molecules that has first and second ends respectively;

(d) each the double-stranded cDNA molecule that derives from step (b) is inserted into into carrier in one direction, it is anti-meaning for a T3 promotor, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', 3 ' flanking vector of 3 ' end of 5 ' the terminal and sense strand that inserts the cDNA sense strand, and described described first restriction endonuclease sites of being structured in of one 3 ' flanking vector sequence and qualification arranged in described promotor, have 15 length of nucleotides between the transcription initiation site at least;

(e) intestinal bacteria that transform with the cDNA insertion carrier that is cut contain the carrier that the clone inserts with generation;

(f) with the sequence at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the structure storehouse that the digestion with restriction enzyme of the sequence in the T3 promotor produces in step (c), so just produced the linear segment of the cDNA molecule that contains insertion.

(g) by incubation linear fragment and a T3RNA polysaccharase that has initial from T3 promoter transcription ability, produced the preparation of the cRNA that antisense cRNA transcribes;

(h) with a ThermoScript II with 15-30 Nucleotide is long and by 5 ' the reverse transcription primer of forming with 5 ' flanking vector sequence complementary nucleotide sequence, produce the first chain cDNA by transcribing cRNA;

(i) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA, the polymerase chain reaction is that 15-30 Nucleotide is long with first 3 ' PCR primer for the first time, and it is at first restriction endonuclease sites with limit by 3 ' flanking vector sequence complementation between the site of T3 specificity promoter transcription initiation, and first 5 ' PCR primer has been defined-N ₁3 ' the end of forming, wherein " N " is among four kinds of deoxyribonucleotide A, C, G or T one, this first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier preface complementation of 5 ' flank, simultaneously, the complementation of first 5 ' PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, in different primers of this first 5 ' PCR primer are used in four inferior storehouses of difference each;

(j) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product, second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, and it is at first restriction endonuclease sites with limit by 3 ' flanking vector sequence complementation between the site of T3 specificity promoter transcription initiation, and second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (i), x is by selecting in 3 and 4 groups that form, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, the simultaneously complementary special insertion Nucleotide that is equivalent to the insertion cRNA of " x "+1 with Nucleotide quantity that extends of primer, at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and to each inferior storehouse, has 4 in the inferior storehouse of second series ^xYa Ku;

49, according to the method for claim 48, the mixture of wherein said 48 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEO ID NO:5).

50, according to the method for claim 48, the mixture of wherein said 48 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:8).

51, according to the method for claim 48, the mixture of wherein said 12 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO:4).

52, according to the method for claim 48, the mixture of wherein said 12 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID No:7).

53, according to 48 methods of claim, the mixture of wherein said 3 anchor primers has the sequence of A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:3).

54, according to the method for claim 48, the mixture of wherein said 3 anchor primers has the sequence of G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO:6).

55, according to the method for claim 48, wherein said first restriction enzyme is MspI, and second restriction enzyme is NcotI.

56, according to the method for claim 48, wherein said first 5 ' PCR primer is G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:22).

57, according to the method for claim 48, wherein said first 3 ' PCR primer and second 3 ' PCR primer are G-A-G-C-T-C-C-A-C-C-G-C-G-T (SEQ ID NO:47).

58, according to the method for claim 48, " x " in the wherein said step (j) is 3.

59, according to the method for claim 48, " x " in the wherein said step (j) is 4.

60, the method for the relation of the variation of a kind of detection mRNA expression pattern in tissue and physiology or pathology variation, it may further comprise the steps:

(a) acquisition is not subjected to first sample of a tissue of physiology or pathology variation;

(b) from first sample, separate a mRNA storehouse;

(c) produce the demonstration of first distinguished sequence product of 3 ' of mRNAs that representative occurs-terminal by (a)-(j) step of carrying out claim 1 in first sample, determine the pattern that mRNA expresses in first sample of tissue;

(d) acquisition is subjected to second sample of a tissue of physiology or pathology variation;

(e) from second sample, separate a mRNA storehouse;

(f) produce the demonstration of second distinguished sequence product of 3 ' of mRNAs that representative occurs-terminal by (a)-(j) step of carrying out claim 1 in second sample, determine the pattern that mRNA expresses in second sample of tissue;

(g) compare first and second demonstrations to determine to change on physiology or the pathology influence to mRNA expression pattern in the tissue.

61, according to the method for claim 60, the variation on wherein said physiology or the pathology is from contacting, reach to contact the process that is mediated between cytolemma and cytoskeleton and screen by basis outside second messenger, hormone, neurotransmitter, somatomedin and neuromodulator, cell-cells contacting, cell-substrate contact, the cell-born of the same parents in transcription factor, the cell.

62,, wherein said tissue-derived in a central nervous system according to the method for claim 60.

63, according to the method for claim 62, the variation on wherein said physiology or the pathology be from by senile dementia, Parkinson's neurological dysfunction, local asphyxia, be addicted to drink, be addicted to drug, screen the group that schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, dysthymia disorders, bipolar manic depressive disorder are formed.

64, according to the method for claim 62, the dispensing of the variation on wherein said physiology or the pathology and study, memory, mood, glutamate dehydrogenase neurotoxic, feed behavior, sense of smell, vision and moving obstacle, virus infection, electroshock or medicine is relevant with the toxic side effect of medicine.

65, according to the method for claim 60, the variation on wherein said physiology or the pathology is from being changed, worn out by circadian rhythm and strengthening the group that is formed for a long time and select.

66, according to the method for claim 60, wherein said tissue be a structure in the central nervous system of from the group that forms by retina, cerebellar cortex, sense of smell bubble, thalamus, hypothalamus, preceding hypophysis, back hypophysis, hippocampus, nucleus accumbens septi, tonsilla, striatum, cerebellum, brain stem, suprachiasmatic nucleus and backbone rope, screening derive and.

67, according to the method for claim 60, wherein said tissue is normal or neoplastic tissue, and it is the organ or the organ-tissue of the screening from the group that is made up of cardiovascular systems, pulmonary system, Digestive tract, peripheral nervous system, liver, kidney, skeletal muscle and reproductive system.

68, according to the method for claim 60, normal or the neoplastic tissue that wherein said tissue is made up of cell, it is that the organ or the organ-tissue of the screening from the group that is made up of neural system, endocrine system, body (being comprised skin, hair and nail), Skeletal system (comprising bone and muscle), urinary system and the reproductive system of cardiovascular systems, lymphsystem, respiratory system, Digestive tract, peripheral nervous system, central nervous system, intestines extracts or deutero-.

69, method according to claim 60, normal or the neoplastic tissue that wherein said tissue is made up of cell, it is from by epithelium, endothelium, mucous membrane, body of gland, blood, lymph, conjunctive tissue, cartilage, bone, unstriated muscle, skeletal muscle, cardiac muscle, neurone, neurogliocyte, spleen, thymus gland, hypophysis, Tiroidina, parathyroid gland, adrenal cortex, adrenal medulla, pineal gland, skin, hair, nail, tooth, liver, the pancreas lung, kidney, bladder, ureter, mammary gland, ovary, the uterus, vagina, Testes, prostate gland, penis, organ that screens in the group of an E ﹠ E composition or organ-tissue extract or deutero-.

70, medicine and a known compound effect diverse ways of a screening of a kind of detection, it may further comprise the steps:

(b) from first duplicate samples, separate a mRNA storehouse;

(c) produce the demonstration of first sequence-specific product of 3 ' of mRNAs that representative occurs-terminal by (a)-(j) step of carrying out claim 1 in first duplicate samples, determine the pattern that mRNA expresses in first sample tissue;

(e) from first duplicate samples, separate a mRNA storehouse;

(f) produce the demonstration of second sequence-specific product of 3 ' of mRNAs that representative occurs-terminal by (a)-(j) step of carrying out claim 1 in second duplicate samples, determine the pattern that mRNA expresses in second sample tissue;

(g) relatively first and second 's demonstration, so that detect the existence of mRNA species, the expression of mRNA is not subjected to the influence of known compound, but is subjected to the influence of screened medicine, therefore, shows the medicine of screening and the difference of known compound effect.

71, according to the method for claim 70, wherein said screened medicine is to select from the group of being made up of thymoleptic, Antipsychotic drug, tranquilizer, anticonvulsive drug, oxidase inhibitor, stimulant.

72, according to the method for claim 70, wherein said screened medicine is to screen from the group that is made up of anti-Parkinson agent, skeletal muscle relaxant, anodyne, toponarcosis, cholinergic agent, antiviral agent, spasmolytic, steroid and non-steroid anti-inflammatory drug.

73, database, it comprises the data by the quantitative generation of the demonstration of the sequence specific product that passes through claim 1.

74, the database of claim 1, it further comprises containing and relates to the data that serial correlation, gene mapping and cell distribute.

75, the sequence identity between a kind of 3 '-end sequence that is identified in the mRNA molecule that occurs in the sample and the sequence library and the method for similarity, it comprises the steps:

(a) prepare a double-stranded cDNA storehouse with a blended anchor primer from a mRNA storehouse, each anchor primer contains one 5 ' terminal and one 3 ' end, and comprises: (i) one of 7-40 T residue section sequence; (ii) discern the site of first restriction enzyme cutting of 6 above bases, this cleavage site is relevant with the sequence of T residue towards 5 '-terminal location; (iii) first stuffer of from 4 to 40 Nucleotide, this first stuffer be positioned toward with by 5 ' relevant-end of first restriction enzyme cleavage site; (iv) second stuffer inserts 6 above bases of identification by between first restriction enzyme cleavage site and the T residue sequence; (v) the phase transformation residue be positioned in from by-V ,-V-N and-3 '-end of each anchor primer of selecting the group that V-N-N forms, wherein V is from forming the deoxyribonucleotide that screens the group by A, C and G; N is the deoxyribonucleotide that screens from the group that is made up of A, C, G and T, and the mixture that includes anchor primer contains the possibility of all V and N;

(b) downcut double-stranded cDNA storehouse with first sex-limited restriction endonuclease and second restriction enzyme, 4-nucleotide sequence of second restriction enzyme identification has formed a double-stranded cDNA library of molecules that first and second ends are arranged respectively.

(c) each the double-stranded cDNA molecule that derives from step (b) is inserted into into carrier in a direction, it is anti-meaning for the special promotor of phage, in carrier, formed and contained the structure storehouse of inserting the cDNA molecule, therefore determine respectively in abutting connection with the sequence of 5 ', the 3 ' flanking vector that inserts the terminal and sense strand 3 ' end of cDNA sense strand 5 ', and the described structure that one 3 ' flanking vector sequence arranged has 15 length of nucleotides in described first restriction endonuclease sites and qualification at least between the transcription initiation site in described promotor;

(e) with the sequence at least a cDNA molecule that maybe can not discern insertion, maybe can not discern the sequence in the special promotor of phage, but the structure storehouse that the digestion with restriction enzyme of sequence produces in step (c) on the energy identification carrier, so just produced the linear segment of the cDNA molecule that contains insertion, the linear fragment of Chan Shenging has a 5 ' flanking vector sequence of holding at least 15 Nucleotide of second end of double-stranded cDNA molecule at carrier 5 ' like this;

(g) with a ThermoScript II with 15-30 Nucleotide is long and by 5 ' the reverse transcription primer of forming with 5 ' flanking vector sequence complementary nucleotide sequence, produce the first chain cDNA by transcribing cRNA;

(h) form the first serial inferior storehouse and carry out the polymerase chain reaction generation first time first cover PCR product as template by separating the first chain cDNA with the first chain cDNA, the polymerase chain reaction is that 15-30 Nucleotide is long with first 3 ' PCR primer for the first time, it and determine 3 ' flanking vector sequence complementation between the transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, first 5 ' PCR primer has been defined-N ₁3 '-end of forming, wherein " N " is among four kinds of thymus nucleic acid A, C, G or T one, first 5 ' PCR primer is that 15-30 Nucleotide is long, and and the carrier preface complementation of 5 ' flank, simultaneously, the complementation of first 5 ' PCR primer extends into a Nucleotide in the special insertion Nucleotide of cRNA, in different primers of this first 5 ' PCR primer are used in four inferior storehouses of difference each;

(i) become the inferior storehouse of second series by the further separation first serial inferior storehouse cover of first in each PCR product and carry out polymerase chain reaction generation second time second with the first cover PCR product as template and overlap the PCR product, second 3 ' the PCR primer that the polymerase chain reaction is used in the second time is that 15-30 Nucleotide is long, it and determine 3 ' flanking vector sequence complementation between the transcription initiation site at first restriction endonuclease sites with by the special promotor of phage, second 5 ' PCR primer has been defined one by-N ₁3 '-end that-Nx forms is at this N ₁With the N that is used in the polymerase chain reaction for the first time for that inferior storehouse ₁Be the same, " N " is as in step (h), x is from 1 to 5 integer, this primer length is a 15-30 Nucleotide, and and 5 ' flanking vector sequence complementation, the complementary special insertion Nucleotide that is equivalent to the insertion cRNA of " x "+1 with Nucleotide quantity that extends of primer simultaneously is at this, the 25 ' each different primer is used in the different inferior storehouse in the inferior storehouse of second series in the PCR primer, and has 4 for each inferior storehouse in the inferior storehouse of first cover in the inferior storehouse of second series ^xYa Ku;

(l) cDNA of amplification wash-out in a polymerase chain reaction;

(m) clonal expansion cDNA enters in the plasmid;

(n) produce with the corresponding DNA of DNA that derives from the clone of plasmid;

(o) sequence of definite clone's cDNA;

(p) determine corresponding nucleotide sequence from a Nucleotide database, the sequence that corresponding nucleotide sequence described here is defined between being begun by the recognition site of second restriction enzyme least significant end and Poly (A) tail;

(q) relatively clone cDNA sequence and corresponding nucleotide sequence, therefore can identify the identity and the similarity of 3 '-end sequence and the database sequence of a present sample mRNA.

76, according to the method for claim 76, it further comprises the steps:

(r) length and the quantity of comparison PCR product in a two-dimensional map shows.

77, according to the method for claim 76, it further comprises the steps:

(s) determine the prediction length of corresponding Nucleotide, this length is identical with the length of a large amount of corresponding nucleotide product of determining from database, determine can with the length of 5 ' PCR sequence of carrier hybridization, determine remaining anchor primer carrier sequence the intervention part length and and the length of interfertile 3 ' the PCR sequence of carrier;

(t) length of the corresponding nucleotide sequence of PCR product length and fixed prediction relatively is certified in a two-dimensional map shows by a graphic indicia or Chinese character in the length of the prediction of this corresponding nucleotide sequence.

78, a kind of identification and the identity between the corresponding cDNA fragment sequence of a mRNA molecule of a sample appearance and sequence library and the method for similarity, it comprises the steps:

The cDNA fragment of clonal expansion enters a plasmid;

Produce a dna molecular of and cDNA fragment correspondence;

Therefore the check order dna molecular of that generation has determined the cDNA fragments sequence of wash-out; And,

Sequence in the cDNA fragments sequence of comparison wash-out and the database, so the identity of recognition sequence and similarity.

79, according to the method for claim 78, the step of sequence is carried out with computer in the cDNA fragments sequence of wherein said relatively wash-out and the database.

80, according to the method for claim 78, it comprises the additional step that shows comparative result with diagram.

81, the sequence identity between a kind of identification and mRNA molecule of appearance is corresponding in a sample cDNA fragments sequence and the database sequence and the method for similarity, it comprises the steps:

Wash-out divides corresponding cDNA fragment with a mRNA who occurs in a sample, the cDNA fragment has a length of being determined by the position of a Poly (A) tail of a restriction endonuclease sites and that mRNA molecule here;

With with one 5 ' PCR primer of that restriction endonuclease sites correspondence, determine the segmental partial sequence of cDNA by the polymerase chain reaction; And

Compare definite partial sequence of wash-out cDNA fragment and cDNA fragment length, the identity and the similarity of coming recognition sequence for sequence in database.

82, a kind of method that produces the registration of a conversion polynucleotide sequence database, it may further comprise the steps:

From a polynucleotide sequence database registration, select a source sequence;

A location poly (A) tail sequence in source sequence;

One of location and the hithermost restriction enzyme enzyme recognition site of first restriction enzyme sequence in source sequence;

In source sequence, determine a relevant sequence, described correlated series comprises the sequence that is defined by poly (A) tail and restriction enzyme enzyme recognition site, and comprises the part of restriction enzyme enzyme recognition site at least;

Determine the length of correlated series; With

Therefore storage can produce the database registration of a conversion about the information of the length of the position of poly (A) tail and sequence, correlated series that restriction enzyme enzyme recognition site position is relevant with source sequence with sequence.

83,2 method according to Claim 8, it further comprises following step:

Diagram shows the length of the correlated series relevant with index sequence.

84,3 method according to Claim 8, wherein said restriction endonuclease sites are from by MspI, TaqI, select in the group that HinP1I forms.

85, a kind of by reducing because the background of non-target cDNAs amplification is improved the method for the resolving power of the length of PCR product and quantity, it comprises the steps:

The sample in a cDNAs storehouse of screening, here each cDNA molecule includes insertion sequence and the sequence that derives from carrier;

Can carry out reverse transcription with the reverse transcription primer of the sequence hybridization that derives from carrier with one, extend about 5-6 Nucleotide and enter the product that insertion sequence produces a cDNA reverse transcription;

The product that separates the cDNA reverse transcription again;

86,5 method according to Claim 8 wherein has the reverse transcription reaction reverse transcription primer different with 16 in 16 storehouses.

87,5 method according to Claim 8 wherein has 4 ^xThe polymerase chain reaction in inferior storehouse, at this, the quantity that 5 ' PCR primer extension enters the Nucleotide of insertion sequence is different with the quantity that the reverse transcription primer extension enters the Nucleotide of insertion sequence.