CN100487117C - Method for quickly separating target chromosome expressed sequence - Google Patents

Abstract

The invention relates to a process for separating expression sequence on destination chromosome, which consists of disintegrating destination chromosome, obtaining destination chromosome DNA with joints, obtaining total cell cDNA with another kind of joints, subjecting the destination chromosome DNA and total cell cDNA to isogenesis hybridization, carrying out inhibited PCR expansion isogenesis sequence, thus obtaining the expression sequence on the destination chromosome. The advantages of the process include short cycle and simplified operation.

Description

A kind of method of quickly separating target chromosome expressed sequence

Technical field

The present invention relates to a kind of method of quickly separating target chromosome expressed sequence.

Background technology

Little cutting of karyomit(e) and microclone (Chromosome microdissection andmicrocloning) are meant at microscopically, by micromanipulation system target chromosome or chromosome segment are cut, target chromosome or chromosome segment are separated, and the clone is positioned at the technology of the DNA on the target chromosome.Its Application Areas is not confined to the cytogenetics level to chromosome research, in chromosome evolution research (Jamilena etc., 1995; Houbean etc., 1996), construction of genetic atlas (Schondelmaier etc., 1993; Arumuganathan etc., 1994), genome physical mapping, molecule marker genetic linkage maps make up (Jung etc., 1992; Schondelmaier etc., 1993), chromosomal in situ hybridization or chromosome painting probe (Zhou etc., 1999; Fu Junjiang etc., 1998) etc. research field also is used widely.Though above several respects work has obtained some achievements, the little cutting of karyomit(e), differential are from the real advantage of technology and underuse.Unless this work and functional genome research are connected.From this angle, the expressed sequence of separate targets karyomit(e) or chromosome segment will make this The Application of Technology more valuable.Because the separation of the expressed sequence on the target chromosome, the molecule marker of mapping mark and gene not only can be provided, also relevant with functional genome research work, and may be the effective ways of functional gene on separate targets karyomit(e) or the chromosome segment.Some relevant work reports have been arranged at present, promptly at the little cutting of karyomit(e) and differential on the basis of technology, further analyze the expressed sequence of corresponding karyomit(e) or chromosome segment.

In recent years, developed multiple method from genomic dna or chromosomal DNA separation expressed sequence.(1) probe sieve method.This is a kind of conventional direct sieve method of probe hybridization that is based on.It mainly contains dual mode, a kind of mode is as probe with the fragment in the karyomit(e) microclone library, screening homology fragment promptly is called direct sieve method again in the cDNA library, and another kind is to utilize the cDNA fragment as probe karyomit(e) microclone library to be screened.This method is that accuracy should be very high, utilizes this method to separate corresponding expressed sequence (Yokoi etc., 1994 from people and animal karyomit(e) at present based on the dot blot of routine; Kao etc., 1994; Wei etc., 1996; Yu etc., 1997).But the main shortcoming of this method relates to loaded down with trivial details steps such as a large amount of clone operations and clone hybridization screening, and is time-consuming.(2) by the cDNA prize law (Microdissection-mediated cDNA capture) of micro-dissections.In this method, be that probe directly carries out in situ hybridization to chromosome sectioning at first with the cDNA that adds joint, by thorough washing the not hybridization cDNA on the chromosome sectioning is washed off then, again by the target fragment on micrurgy cutting and separate targets karyomit(e) or the karyomit(e), the target chromosome fragment that is separated to is as template, carry out the pcr amplification of stringent condition with the primer on the cDNA joint, the cDNA of the homology of DNA hybridization just is amplified (Yokoyama etc., 1997 on those and this karyomit(e) as a result; Kim etc., 2001; Gracia etc., 1997).This method complex operation directly causes the isolating efficient of homology segment.Compare with above method, using method of the present invention is in liquid-phase operation, and step is simple, and late detection is accurately quick, and false positive rate is low, expressed sequence separation efficiency advantages of higher.

Summary of the invention

At above-mentioned research background, inventor's further investigation, adopt the little cutting of karyomit(e), microclone technology, simultaneously with homologous fragment specific amplified technology (Hybrid specific amplification, HSA, Lecerf F etc. 2001) combine, and with reference to suppressing subtractive hybridization (suppressionsubtractive hybridization, SSH, Diatchenko L. etc., 1996) relative theory in the technology and program have been set up the method for a quickly separating target chromosome expressed sequence.

An object of the present invention is to provide the method for the chromosomal expressed sequence of a kind of separate targets, comprise following steps:

(1) separate targets karyomit(e);

(2) acquisition has the target chromosome DNA of joint;

(3) acquisition has total cell cDNA of another kind of joint;

(4) above-mentioned target chromosome DNA and total cell cDNA are carried out homology hybridization;

(5) suppress the pcr amplification homologous sequence, obtain the expressed sequence on the target chromosome.

In one embodiment of the invention, obtain target chromosome by the isolating method of little glass needle.

In another embodiment of the invention, the acquisition of target chromosome DNA is that the method (LA-PCR or DOP-PCR method) of utilizing PCR obtains, and the target chromosome DNA that obtains is cut with the restriction endonuclease enzyme, and add jointing.

In another embodiment of the invention, the acquisition that has the cDNA of another kind of joint is that total cell mRNA reverse transcription of will extract becomes cDNA, and adds top connection.

In another embodiment of the invention, the acquisition of expressed sequence is to utilize strand cDNA or double-stranded cDNA on the target chromosome.

In a preferred embodiment of the present invention, the acquisition of expressed sequence comprises that utilizing strand cDNA to carry out target chromosome expressed sequence separates on the target chromosome, and it comprises:

(1) utilizes 3 ' terminal specific primer reverse transcription, first chain, obtain the different cDNA of length;

(2) merge the sex change repeatability with target chromosome DNA, add 100 times of Cot1DNA and blockade, form the hybridization chain of chromosomal DNA and cDNA;

(3) utilize 3 ' the terminal specific primer of joint sequence on the chromosomal DNA and cDNA to suppress pcr amplification hybridization chain, obtain described chromosomal expressed sequence.

In another preferred embodiment of the present invention, the acquisition of expressed sequence comprises that utilizing double-stranded cDNA to carry out chromosome expressed sequence separates on the target chromosome, and it comprises:

(1) reverse transcription synthetic double chain cDNA, the restriction endonuclease enzyme is cut, and adds jointing;

(2) DNA with target chromosome or chromosome segment merges the sex change repeatability, adds 100 times of Cot1DNA and blockades, and forms the hybridization chain of chromosomal DNA and cDNA;

(3) utilize the joint sequence of joint sequence on the chromosomal DNA and cDNA to suppress pcr amplification hybridization chain, obtain chromosome expressed sequence.

Owing to used the DNA reannealing kinetics in the method for the present invention and suppressed the PCR principle, can reduce high copy number of fragments among chromosomal DNA and the cDNA, and can avoid being subjected in little cutting chromosomal DNA amplification procedure the pollution of foreign DNA, can also enrichment the dna fragmentation of low expression amount, for important foundation has been established in the separation of target chromosome EST and new gene.

Description of drawings

The present invention is described in further detail below in conjunction with the drawings and specific embodiments, is that the present invention is limited but should not be construed as.

Fig. 1 utilizes strand cDNA to separate the technological line of chromosome expressed sequence;

Fig. 2 utilizes double-stranded cDNA to separate the technological line of chromosome expressed sequence;

Fig. 3 is the chromosomal micro-sepn process of rye 1R (black arrow is depicted as the rye 1R karyomit(e) position that has satellite among the figure, and the white arrow indication is for separating and sticking to 1R karyomit(e) on the glass needle);

Fig. 4 is the electrophoresis result (1.0% sepharose) of the amplified production of chromosomal DNA;

Fig. 5 is the electrophoresis result (1.5% sepharose) of 1R chromosomal DNA homologous expressed sequence amplified production, 1: directly use PN1 amplified hybridization end product result; 2: directly use PN2 amplified hybridization end product result; 3: two of hybridization end product is taken turns the pcr amplification result; 4: molecular weight marker (be followed successively by from top to bottom 1000,750,500,250,100bp);

Fig. 6 is 1R chromosomal DNA homologous expressed sequence clone's a PCR product electrophoresis;

Fig. 7 is hybridized specific amplified end product clone's dot blot analysis

A. be the dot blot result of probe with rye total cDNA in seedling stage

B. with the dot blot result of the 1R chromosomal DNA amplified production probe of micro-dissections

C. with the rye genome DNA dot blot result of probe, wherein 8A is 1R DNA, 8B is cDNA, 8C is the rye genomic dna, 8D is the Chinese spring genomic dna, and 8E is and separates the end product that obtains, i.e. homologous sequence fragment among 1R chromosomal DNA and the cDNA, 1H is blank plasmid DNA, and other inserts segmental amplified production for clone's that obtains with primer PN1 and PN2 amplification.

Embodiment

To achieve these goals, a kind of preferred implementation of the present invention is as follows.

Micro-separate targets karyomit(e), (Johnson etc. 1990 for method for LA-PCR (linker adaptor PCR) or DOP-PCR (degenerate oligonucleotide primed PCR); Wesley etc., 1989; Telenius etc., 1992) obtain chromosomal DNA, wherein preferred micro-separation is to use little glass needle to separate purpose karyomit(e) by inverted microscope.Isolating karyomit(e) directly put into contain the Proteinase K reaction buffer, concentration is 50ng/ μ l, with 1 * T ₄The preparation of dna ligase damping fluid.Incubation in 37 ℃ of water-baths is so that Proteinase K fully digests Deproteinization, and the time is 4 hours, and 70 ℃ of incubation 20min make enzyme deactivation then.If adopt the method for DOP-PCR, can put into-20 ℃ standby.If adopt the LA-PCR method, then in former system, add restriction endonuclease Sau3A-I and carry out enzyme and cut, consumption is 0.02U, and the reaction times is 3 hours, and 70 ℃ of 20min make enzyme deactivation.Adding the concentration for preparing is the joint 2 μ l of 5ng/ μ l, and use therein joint is the Sau3A joint, with 1 * T ₄Ligase enzyme damping fluid and 20mmol/L ATP are mixed with the working concentration of 5ng/ μ l.The Sau3A joint is that two oligonucleotide fragments by 23mer and 19mer form, during the preparation joint, earlier with 23mer oligonucleotide phosphorylation, with etc. mole 19mer oligonucleotide co-variation annealing make.Add T ₄Dna ligase 0.5 μ l (3U/ μ l), the ligation final volume is 24.5 μ l.After 16 ℃ of connections are spent the night, 70 ℃ of 20min inactivation ligase enzymes.At separating chromosomal source, grope the protease K digesting albumen time, with the common wheat material, digest and be advisable in 4 hours.

The karyomit(e) of above-mentioned processing is carried out two-wheeled LA-PCR amplification or DOP-PCR amplification acquisition chromosomal DNA.LA-PCR first round substrate is the chromosomal DNA that has joint, is that primer carries out 35 cyclic amplifications with the 19mer of corresponding Sau3A joint.Second to take turns amplification be that to get 5 μ l first round products be substrate, 25 circulations of increasing.DOP-PCR first round substrate is the karyomit(e) of handling through Proteinase K, carries out 35 cyclic amplifications with degenerated primer.Second takes turns amplification, and to get 5 μ l first round products be substrate, 25 circulations of increasing.Wherein, in order effectively to avoid the pollution of foreign DNA, above steps guarantees to carry out under aseptic condition as far as possible.Slide glass and cover glass are in advance at washing lotion or hydrochloric acid: soak among ethanol=1:9 more than one day, place 95% ethanol after distilled water is rinsed well again, dry, 120 ℃ of bakings 2 hours are stand-by.Suction pipe, filter paper, blade are all through autoclaving, and the carbol fuchsin dye liquor is after the suction filtration sterilization, and ultraviolet lamp irradiation down carried out compressing tablet more than 1 hour in Bechtop.Eppendorf pipe, the first-class autoclaving that all passes through of Tip that pcr amplification uses, uviolizing is handled more than the 30min again.Solution such as enzyme liquid such as sterilized water, enzymolysis mixed solution, manual splice, Proteinase K are all handled through suction filtration or ultraviolet disinfection.

Said process is established two kinds of strict contrasts: it is initial substrate that positive control adds about 10pg genomic dna, does not add any substrate in the negative control, and other all operations process is the same.This is an indispensable step of monitoring test effect and pollution condition.

Hybridization pre-treatment chromosomal DNA.Merge an amount of second and take turns the PCR product in centrifuge tube, add equal-volume phenol/chloroform, centrifugal 1 minute of 11000g, use equal-volume phenol/chloroform extracting again after getting supernatant, supernatant with the extracting of chloroform the same manner is once got and is reset and added 1/10 volume 3MNaAc (pH5.8) after the extracting, twice ethanol precipitates 2 hours in-20 ℃, centrifugal 10 minutes of 16000g, 70% ethanol is washed once, is dissolved in after the drying at room temperature in the 10 μ l deionization sterilized waters.In 50 μ l systems, cutting 37 ℃ of enzymes of chromosomal DNA (1 μ g) with 10U Sau3A enzyme cuts and spends the night, 70 ℃ of deactivation 20min, phenol/chloroform extracting is behind the ethanol sedimentation, be dissolved in (300ng/ μ l) in an amount of deionization sterilized water, add 1 μ l joint (joint one), joint concentration 10 μ M, joint adopt subduing of Clontech company to suppress the hybridization kit center tap, in 10 μ l systems, add 0.5U T ₄Dna ligase (New England Biolabs), 16 ℃ connect 16 hours, 72 ℃ of deactivation 10min ,-20 ℃ of storages are standby.When utilizing the LA-PCR method to obtain chromosomal DNA, the joint that uses 23mer and 19mer sequence to make is a primer with 19mer, and this primer annealing temperature is lower, can improve amplification efficiency.But in the inhibition PCR of back, to suppress effect and improve specific amplification in order to reach, cut so chromosomal DNA is carried out the secondary enzyme, changing joint is joint one.

CDNA prepares.Positive plant RNA after extraction certain period positive plant or the process special processing, if utilize strand cDNA to separate chromosome expressed sequence, synthetic use 5 ' the connection M of first chain _4-13The Oligo-dT of sequence is a primer, the synthetic back of first chain purifying, and it is standby to dilute about 300ng/ μ l.If utilize double-stranded cDNA to separate chromosome expressed sequence, then continue the synthetic second chain cDNA and carry out end-filling.The double-stranded cDNA of purifying is dissolved in the appropriate amount of deionized water, in 50 μ l systems, spend the night 70 ℃ of 20 minutes deactivation restriction enzymes with 10-15U restriction endonuclease Sau3A digestion, pass through phenol/chloroform purifying then, ethanol sedimentation is dissolved in (300ng/ μ l) in the appropriate amount of deionized water, adds 1 μ l joint (joint two), joint concentration 10 μ M, joint adopts subduing of Clontech company to suppress the hybridization kit center tap, in 10 μ l systems, adds 0.5U T ₄Dna ligase (New England Biolabs), 16 ℃ connect 16h, 72 ℃ of deactivation 10min ,-20 ℃ of storages are standby.

Expressed sequence on the separate targets karyomit(e).

Homology hybridization.With synthetic strand or the about 300ng of double-stranded cDNA, the about 500ng of addition chromosome amplified production that connects good joint one and 100 times the common ethanol sedimentation of Cot1, be dissolved in 4 μ l hybridization buffer (50mM Hepes, pH8.3; 0.5M NaCl; 0.02mM EDTA, pH8.0; 10% (wt/vol) PEG 8000), on cover paraffin oil, 98 ℃ of sex change 1.5min, 68 ℃ hybridization 10 hours.Add fresh strand cDNA or the about 300ng of double-stranded cDNA (being dissolved in 1 * hybridization buffer) after 10 hours, continue under similarity condition, to hybridize 10 hours.Hybridization finishes back dilution buffer liquid (20mM Hepes, pH8.3; 50mM NaCl; 0.2mM EDTA) be diluted to 100 μ l, 72 ℃ add thermally-stabilised 7 minutes ,-20 ℃ of storages.

Suppress PCR.At PE480 (Perkin Elmer, Norwalk, Conn., USA) carry out on, the two-wheeled reaction is 25 μ l systems, and program is with Clontech subtractive hybridization PCR program (PCR-Select cDNA Subtractive Kit), wherein, if utilize strand cDNA to separate chromosome expressed sequence, the first round primer P1 and M _4-13, second takes turns with PN1 and M _4-13Amplification.If utilize double-stranded cDNA to separate chromosome expressed sequence, the first round primer P1 and P2, second takes turns with PN1 and PN2 amplification.The two-wheeled program all adds single primer amplification and does contrast.

The expressed sequence screening.Get the karyomit(e) second of purifying and take turns PCR product 1 μ l (about 100ng/ μ l), 1 μ l T ₄Dna ligase damping fluid, 1 μ lPGEM-T carrier (promega) (60ng/ μ l), T ₄Dna ligase (New England Biolabs) 1 μ l (3U/ μ l), sterilized water are mended to final volume 10 μ l, mixing, and 4 ℃ connect 16 hours.Transformed into escherichia coli is chosen the white colony of acquisition, and ordinary method is extracted plasmid, with primer PN1 and M _4-13Or PN1 and the PN2 evaluation of increasing, insert fragment and really be these two fragments that primer amplification obtains.The cloned sequence that pcr amplification discharges is respectively got 1 μ l PCR product point at Hybond ⁺On the nylon membrane (Amersham company), three films of parallel point, with separating chromosomal DNA, the genomic dna that the cDNA of positive plant and enzyme are cut is probe and film hybridization respectively.Behind the some film, DNA faced up to be successively placed on the filter paper saturated in the following solution: alkaline denaturation liquid (1.5mol/L NaCl, 0.5mol/L NaOH) sex change 5 minutes; Neutralizer (3mol/LNaCl, 0.5mol/L Tris HCl, pH7.4) 8 minutes; 2 * SSC 5 minutes.The room temperature end is done, and DNA faces down, and (BIO-RAD USA) goes up irradiation 1 minute, hybridizes, and the mark of probe and Southern crossover process are with reference to " the The Guide Bookto DIG System User " of B.M. company at ultraviolet transilluminator.Choose the male cloning and sequencing.

Annotate: primer and joint explanation

1. chromosomal DNA obtains the joint of use, is made by 23mer and 19mer primer, uses 19mer as primer when LA-PCR increases.When DOP-PCR, use 6MW to be primer.

Sequence is as follows:

The 23mer primer: 5 '-GATCCTGAGCTCGAATTCGACCC-3 '

The 19mer primer: 5 '-GGGTCGAATTCGAGCTCAG-3 '

23mer and 19mer synthetic linker are as follows:

5′-GATCCTGAGCTCGAATTCGACCC-3′

3′-GACTCGAGCTTAAGCTGGG-5′

The 6MW primer: 5 '-CCGACTGATCNNNNNNATGTGG-3 ' (N=A, T, C, G)

2. karyomit(e) uses primer and joint when hybridizing

Joint one,

5′-GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′

3′-CCCGTCCACTAG-5′

P1，5′-GTAATACGACTCACTATAGGGC-3′

PN1，5′-TCGAGCGGCCGCCCGGGCAGGT-3′

3. cDNA uses joint when hybridizing

Joint two,

5′-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3′

3′-CCTCCCGCCACTAG-5′

P2，5′-TGTAGCGTGAAGACGACAGAA-3′

PN2，5′-AGGGCGTGGTGCGGAGGGCGGT-3′

4. strand cDNA5 ' connection M4-13 sequence

M13-4，5′-GTTTTCCCAGTCACGAC-3′

Embodiment

The separation of rye 1R chromosome expressed sequence

One material and method

1. material and chromosome specimen preparation

Material is rye (S.cereale L) (Chinese Academy of Sciences's heredity provides with growth institute).Rye tool megachromosome is about 8～14 μ m, mainly is made up of tool middle part and nearly metacentric chromosome, and 2n=14 wherein has only a pair of 1R karyomit(e) to have satellite.

Chromosome specimen preparation: 1) the rye seed is sprouted in greenhouse (25 ℃), when root grows to 0.5～1cm, be can be used for pre-treatment.The seed that processing is sprouted in 0～4 ℃ frozen water 24 hours can obtain enough metacinesis phases.2) pretreated germinating seed is placed 95% ethanol: the Kano stationary liquid of Glacial acetic acid (3:1) is fixed 10 minutes, goes in 70% ethanol again, and 4 ℃ of preservations are stand-by.3) seed is cleaned in the distilled water of sterilization, cut the tip of a root.With the tip of a root place 2% cellulase (Onozuka, R-10, Serva) with 2% polygalacturonase (Y-23, in mixed solution Serva), 37 ℃ of enzymolysis 1 hour; It is inferior to give a baby a bath on the third day after its birth with sterile distilled water afterwards, carbol fuchsin dyeing compressing tablet, microscopy.Liquid nitrogen freezes the sheet method and removes cover plate, is used for the micro-dissections operation after direct or gas is done.

2. primer and joint

1) LA-PCR that is used for chromosomal DNA amplification uses primer and joint: with reference to 2 primers of people such as Albani (1993) design:

The 23mer primer: 5 '-GATCCTGAGCTCGAATTCGACCC-3 ';

The 19mer primer: 5 '-GGGTCGAATTCGAGCTCAG-3 ',

After primer 1 phosphorylation, add primer 2,90 ℃ of sex change 2min, 55 ℃ of annealing 20min are cooled to room temperature and become joint.

2) be used for the degenerated primer that chromosomal DNA increases: this primer is to design on degenerated primer 6MW (Telenius etc., 1992) basis.Sequence is the DOP-primer:

5′-CCGACTGATCNNNNNNATGTGG-3′

3) be used to hybridize the joint of sample

Joint one, 5 '-GTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3 '

3′-CCCGTCCACTAG-5′

Joint two, 5 '-TGTAGCGTGAAGACGACAGAAAGGGCGTGGTGCGGAGGGCGGT-3 '

3′-CCTCCCGCCACTAG-5′

Above-mentioned joint and primer are all to be joint and the primers (Diatchenko etc., 1996) that are applied among the SSH, but joint is changed into the GATC cohesive end of Sau3A by original flush end.

4) amplification PCR primer

P1，5′-GTAATACGACTCACTATAGGGC-3′

P2，5′-TGTAGCGTGAAGACGACAGAA-3′

PN1，5′-TCGAGCGGCCGCCCGGGCAGGT-3′

PN2，5′-AGGGCGTGGTGCGGAGGGCGGT-3′

M _13-4，5′-GTTTTCCCAGTCACGAC-3′

5) joint preparation

The oligonucleotide fragment that will be used for synthetic linker is with the dissolving of TE solution, waits mole to mix post-heating to 70 ℃, at 2 hours internal cooling to 10 ℃.-20 ℃ of preservations.

The pcr amplification of chromosomal micro-separation of 3 rye 1R and DNA thereof

1) the micro-separation of karyomit(e):

Thin glass stick about the 1mm of cut-off footpath, being drawn into diameter on miniature alcohol lamp is the glass needle of 1～3 μ m needle point, the camber that while needle point tool is about 120 ℃.The film-making of choosing is placed the liquid nitrogen quick-frozen several seconds, take out the back and take cover plate rapidly off with blade, gas is done stand-by.The glass needle that makes is fixed on the Leitz micromanipulator (Germany), puts the dried film-making of gas simultaneously under inverted microscope, chooses required division phase under the mirror.Low power lens is pin down slowly down, changes high power lens step by step, regulates the position of needle point, last tip alignment want isolating karyomit(e) come clawback from, it is come off from divide mutually, and sticks on the glass needle point.Slowly, will be stained with chromosomal needle point afterwards and fracture in the PCR pipe of the 20 μ l that contain 10 μ l reaction buffers, the high speed centrifugation several seconds, deposit stand-by for-20 ℃ by micromanipulator lifting glass pin.

Reaction buffer contains 50ng/ μ l Proteinase K (BM) with 1 * Taq damping fluid (Promage) preparation.

2) differential is from chromosomal amplification in vitro

(1) enzymolysis Deproteinization:

Chromosomal PCR pipe is housed is put in 70 ℃ of item for disposal 20min inactivating protein enzyme K behind 37 ℃ of enzymolysis 4h.

(2) enzyme is cut and added joint: adding restriction endonuclease Sau3A-I carries out enzyme and cuts, and consumption is 0.02U, and in 3 hours reaction times, 70 ℃ of 20min connect enzyme deactivation, add Sau3A joint 2 μ l (5ng/ μ l), T ₄Dna ligase 0.5 μ l (3U/ μ l), the ligation final volume is 24.5 μ l.After 16 ℃ of connections are spent the night, 70 ℃ of 20min inactivation ligase enzymes.

(3) pcr amplification:

Add reacted constituent by following system:

10 * Taq enzyme buffer liquid, 5.0 μ l, 2.5mmol/L MgCl ₂2.5mmol/L dNTPs 4.0 μ l, primer (for the LA-PCR primer is the 19mer primer, is degenerated primer 6MW for the DOP-PCR primer) 0.7 μ l (330ng/ μ l), Taq enzyme 0.5 μ l (5U/ μ l), the sterilized water of handling with ultraviolet is mended to final volume 50 μ l mixings at last.

PCR carries out on Biometra PCR instrument (Germany), and the DOP-PCR program is as follows: first round PCR program is: 94 ℃ of pre-sex change 5min, then enter 94 ℃ of 1min, and 30 ℃ of 1.5min, 72 ℃ of 3min, wherein 30 ℃ are changed to 72 ℃ of need 3min.Then enter 25 circulations after 5 such circulations: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1.5min, and every circle extends 1s automatically; Last 72 ℃ are extended 10min, 4 ℃ of insulations.The LA-PCR program is as follows: 94 ℃ of pre-sex change 5min, then enter 94 ℃ of 1min, and 55 ℃ of 1.5min, 72 ℃ of 3min, 35 circulations, last 72 ℃ are extended 10min, 4 ℃ of insulations.

Second to take turns pcr amplification be to get the first round PCR product of 1/10 (5 μ l) as template, and reaction system is the same, and the PCR program is 94 ℃ of pre-sex change 5min; Then 25 circulations: 94 ℃ of 1min, 55 ℃ of 1.5min, 72 ℃ of 3min; Last 72 ℃ are extended 10min, 4 ℃ of insulations.

It is still very weak to take turns amplified signal as electrophoresis detection second, can carry out the third round amplification.Promptly get 1/10 second take turns the PCR product as template, other conditions are taken turns amplification with second.

(3) chromosomal DNA amplification purification:

Merge the PCR product in centrifuge tube, add equal-volume phenol/chloroform, centrifugal 1 minute of 11000g uses equal-volume phenol/chloroform extracting after getting supernatant again, and supernatant with the extracting of chloroform the same manner once after the extracting, get and reset and add 1/10 volume 3M NaAc (pH5.8), twice ethanol in-20 ℃ the precipitation 2 hours, centrifugal 10 minutes of 16000g, 70% ethanol is washed once, add the dissolving of 10 μ l deionization sterilized waters after the drying at room temperature, be stored in-20 ℃.

4. rye RNA in seedling stage separation and cDNA are synthetic

1) the TRIzol method extract total RNA (TRIzol Kit, GIBCOL), undertaken by the description of product:

50-100mg sample+1mL TRIzol grinds to form homogenate; 15-30 ℃ of temperature bathed 5min, adds the 0.2mL chloroform, seals lid, and with the hand 15s that commoves, 15-30 ℃ of temperature temperature bathed 2-3min, and low-temperature centrifugation 15min (＜12,000xg); On move into new pipe mutually, add 0.5mL isopropanol precipitating RNA, 15-30 ℃ of temperature bathed 10min, centrifugal 10min (＜12,000xg); Go phase, add 75% ethanol (〉 1mL) the outstanding precipitation of washing in whirlpool, low-temperature centrifugation 5min (＜7,500xg) do or vacuum is drained (avoid traditional vacuum, do not parch), and the rifle head is blown and beaten up and down and is dissolved in the water that 180 μ l do not contain Rnase by gas.

2) cDNA is synthetic

(1) the synthesizing single-stranded cDNA of reverse transcription reaction (Takara RNA PCR kit (AMV) ver 2.1)

A forms the modulation reaction solution by following reaction

MgCl ₂ 4μl

10 * RNA PCR damping fluid, 2 μ l

The ddH that does not contain Rnase ₂O 8.5 μ l

DNTP mixture 2 μ l

Rnase inhibitor 0.5 μ l

AMV reversed transcriptive enzyme 1 μ l

Oligo dT-joint primer 1 μ l

Sample (the total RNA of 1 μ g) 1 μ l

20 μ l/ samples altogether

Product

B carries out reverse transcription reaction by following condition

42℃ 15min～30min

99℃ 5min

5℃ 5min

Through phenol/chloroform extracting, behind the ethanol sedimentation, be dissolved in (~ 300ng/ μ l) in the deionization sterilized water ,-20 ℃ of storages are standby.

(2) double-stranded cDNA is synthetic

Utilize the double-stranded expressed sequence that separates, must be to total RNA purifying, mRNA separates the mRNA separating kit separation and purification of adopting Stratagene company, and program is as follows:

(i) 65 ℃ of heating RNA sample 5min put on ice, add 20 μ l, 10 * sample buffer make the sample buffer final concentration reach 1 *.Preheating elutriant to 65 ℃;

(ii) take off the purification column two end cap, pull out handle in the 10ml syringe, syringe is connected with purification column, the damping fluid of storing is released with per two seconds one speed;

(iii) take off syringe, in purification column, add 200 μ l high-salt buffers, release high-salt buffer up to emptying with per two seconds one speed.Repeat (iii);

(iv) the RNA sample is added purification column, release in the 1.5ml centrifuge tube that a DEPC handled with per two seconds speed of one.Again cross post by (iv) step with crossing sample that post reclaims;

(v) in purification column, add 200 μ l high-salt buffers, release high-salt buffer, repeat (v) with the speed of one of per second;

(vi) give a baby a bath on the third day after its birth time each 200 μ l with the speed of one of per second with low salt buffer;

(vii) be preheating to 65 ℃ elutriant with the speed eluted rna of one of per second with 200 μ l, the 1.5ml centrifuge tube of handling with DEPC reclaims, and puts on ice;

(viii) ultraviolet spectrophotometer surveys 260 and the reading of 280nm, and 260/280 value is 2 o'clock, and sample is very pure;

(ix) add 500 μ l precooled ethanol in sample ,-20 ℃ are spent the night;

(x) use refrigerated centrifuge with 12000rpm, 4 ℃ of centrifugal 30min, 70% ethanol is washed precipitation, after the precipitation drying, is dissolved in the 20 μ l DEPC treated waters.

Get mRNA 2 μ g, with OligoD (T) Primer carry out cDNA synthetic (ZAP-cDNASynthesis kit, Stratagene).

A.cDNA first chain is synthetic

(i) 37 ℃ of water-baths of preheating;

(ii) melt the first chain reagent of all non-enzymes on ice, outstanding, the centrifugal medicine that makes in brief whirlpool is compiled in the pipe end;

(iii) set up following system (in order) on ice: 5.0 μ l, 10 * Buffer, 3.0 μ ldNTP Mix, 2.0 μ l Link-Primer (1.4 μ g/ μ l), 17.5 μ l DEPC-H ₂O, 1.0 μ l Rnase Block Ribonuclease Inhibitor (40U/ μ l);

(iv) mixing, centrifugal slightly 5s adds and put the mRNA of sex change 5min on ice immediately behind 70 ℃ of water bath heat preservation 15min, mixing gently, 10min anneals under the room temperature;

(v) adding 1.5 μ l MMLV-RT (50U/ μ l) final volume is 50 μ l;

(vi) mixing gently, the centrifugal slightly pipe end that is compiled in;

(vii) 37 ℃ of temperature are bathed 1-1.5h;

(viii) prepare 16 ℃ of water-baths;

(ix) behind the 1h, from 37 ℃ of water-baths, take out first chain reaction and put on ice.

B.cDNA second chain is synthetic

(i) melt the second chain reagent of all non-enzymes on ice, outstanding, the centrifugal medicine that makes in brief whirlpool is compiled in the pipe end;

(ii) order adds in the first chain reaction system: 20 μ l, 10 * Buffer, 6.0 μ l dNTPMix, 114 μ l DEPC-H ₂O, 2.0 μ l Rnase H (1.5U/ μ l) and 11 μ l DNA polymeraseI (9.0U/ μ l);

(iii) mixing gently, the centrifugal slightly pipe end that is compiled in,, 16 ℃ of temperature are bathed 2.5h, notice that temperature is greater than 16 ℃ scarcely, otherwise easily form hairpin structure;

(iv) behind the second chain reaction 2.5h, put on ice immediately.

The C.cDNA end-filling

(i) add following reagent on ice: 23 μ l dNTP Mix, 2 μ l Pfu DNA polymerase (2.5U/ μ l);

(ii) FAST VORTEX outstanding brief centrifugal after, 72 ℃ of temperature are bathed 30min, surpass 30min;

After (iii) reaction finishes, add 200 μ l phenol/chloroforms [1:1 (v:v)] extracting once, room temperature high speed centrifugation 2min, supernatant change another new centrifuge tube over to;

(iv) take out body once, change supernatant in another new pipe with the equal-volume chloroform;

(v) add 20 μ l 3M NaAc, 400 μ l, 100% ethanol, mixing ,-20 ℃ are spent the night;

(vi) 4 ℃, the centrifugal 1h of 12000rpm collects the cDNA precipitation, and 70% ethanol is washed gently, and mixing and whirlpool are outstanding;

(vii) centrifugal 2min at full speed, sucking-off ethanol dries up precipitation;

(viii) precipitation is resuspended in the 10 μ l aseptic deionized waters, and-20 ℃ standby.

5. utilize solution hybridization and PCR method to obtain cDNA fragment on the rye 1R karyomit(e)

1) hybridization specimen preparation

Get each 1 μ g of chromosomal DNA and double-stranded cDNA, cut in 37 ℃ of enzymes with the Sau3A enzyme (Promage) of 10U respectively at 50 μ L systems and spend the night.70 ℃ of 20 minutes deactivation restriction enzymes.Get enzyme and cut product (about 300ng), 1 μ l joint, joint concentration 10 μ M, joint adopt subduing of Clontech company to suppress hybridization kit (Cat.No.637401) center tap, in 10 μ l systems, add 0.5U T ₄Dna ligase (New England Biolabs), 16 ℃ connect 16h, 72 ℃ of deactivation 10min ,-20 ℃ of storages are standby.Wherein chromosomal DNA connects with joint one, and the cDNA fragment connects with joint two.After connection finishes, add 100 times of Cot1DNA common ethanol sedimentation after chloroform/phenol extracting in the chromosomal DNA.When utilizing strand cDNA to separate expressed sequence, in order to obtain 3 ' client information, do not carry out enzyme and cut, only chromosomal DNA is carried out enzyme and cut, all the other are handled with double-stranded.

2) hybridization

With the synthetic strand or added the about 300ng of double-stranded cDNA in street corner, the about 500ng of addition chromosome amplified production that connects good joint and 100 times the common ethanol sedimentation of Cot1, be dissolved in 4 μ l hybridization buffer (50mM Hepes, pH8.3; 0.5M NaCl; 0.02mM EDTA, pH8.0; 10% (wt/vol) PEG8000), cover paraffin oil, 98 ℃ of sex change 1.5min, 68 ℃ of hybridization 10h on.Add fresh strand cDNA or the about 300ng of double-stranded cDNA (being dissolved in 1 * hybridization buffer) behind the 10h, continue under similarity condition, to hybridize 10h.Hybridization finishes back dilution buffer liquid (20mMHepes, pH8.3; 50mM NaCl; 0.2mM EDTA) be diluted to 100 μ l, 72 ℃ add thermally-stabilised 7 minutes ,-20 ℃ of storages.

3) pcr amplification of hybridization sample

Carry out on PE480, two-wheeled reaction is 25 μ l systems, and program is with Clontech subtractive hybridization PCR program, and wherein, if utilize strand cDNA to separate chromosome expressed sequence, first round primer is with P1 and M _4-13, second takes turns with PN1 and M _4-13Amplification.If utilize double-stranded cDNA to separate chromosome expressed sequence, the first round primer P1 and P2, second takes turns with PN1 and PN2 amplification.The two-wheeled program all adds single primer amplification and does contrast, sees the inhibition effect.Template is that 1 μ L has diluted the hybrid dna sample, each 1 μ L (5 μ M), 22 μ L PCR master mixture prepared usingPremix of outside primer ^EX(Takara) the .PCR program is: 72 ℃ of end-filling 10min, 30 PCR circulation (94 ℃ 30 seconds, 68 ℃ 30 seconds, 72 ℃ 1.5 minutes).72 ℃ were extended 7 minutes.Amplified production carries out 15-25 round-robin amplification as template by the PCR condition of the first round with 10 times of TE damping fluid dilutions, the PCR product of getting 1 μ L dilution, and primer is then used the primer of joint inboard instead.

6. the clone of amplified production

Amplified production utilizes T-vector, and (transformant extracts plasmid for T-, Promage) clone, with primer PN1 and M _4-13Or PN1 and the PN2 evaluation of increasing, insert fragment and really be these two fragments that primer amplification obtains.

1) with the ligation of carrier

The carrier that this experiment is adopted is the pGEM-T carrier of buying from Promega company.Because the outstanding T (thymidine) of the even tool of the insertion site 3 ' of this carrier end, can improve greatly its with the PCR product be connected effect (some Taq polysaccharase be everlasting 3 ' end of amplified fragments add an A (adenosine) who has nothing to do with template).Simultaneously the PGEM-T carrier is also had a multiple clone site, has made things convenient for and has inserted segmental release.When carrying out ligation, get karyomit(e) PCR product 1 μ l (about 100ng/ μ l), the 1 μ lT of purifying ₄Dna ligase damping fluid, 1 μ l PGEM-T carrier (60ng/ μ l), T ₄Dna ligase 1 μ l (3U/ μ l), sterilized water are mended to final volume 10 μ l, mixing, and 4 ℃ connect 16h.

2) preparation of competent escherichia coli cell

Activation E.coli bacterial strain DH5 α (Chinese Academy of Sciences's heredity provides with growth institute).Picking list bacterium colony is in 10ml LB liquid nutrient medium from flat board, overnight incubation on 37 ℃ of shaking tables.Next day, get 1ml bacterium liquid and join in the fresh LB substratum of 100ml 37 ℃ of quick wave and culture 2～3h.When D reaches 0.5～0.6 when the OD value, ice bath bacterium liquid 30min.The centrifugal 5min of 6000rpm collects thalline.Abandon supernatant, add the aseptic CaCl of 10ml precooling ₂(0.1mol/L) solution suspension cell.Ice bath 15min.The centrifugal 5min of 6000rpm.Abandon supernatant, somatic cells is suspended in again the CaCl of 2ml ₂(0.1mol/L) in the solution.Divide to install in the Eppendorf pipe of precooling 200 μ l/ pipe.

3) conversion of ligation product

Get connection product 1 μ l in 200 μ l competent escherichia coli cells, behind the abundant mixing, ice bath 30min.42 ℃ of water-bath thermal shock 90s put 1～2min in the ice bath immediately.Add 800 μ l LB liquid nutrient mediums, 1 hour (can place on the shaking table of 37 ℃ of water-baths or 37 ℃ of 150rpm) of 37 ℃ of insulations.On the LB solid plate of Amp (100g/L), evenly be coated with 40 μ l X-gal (20 μ g/ μ l) and 4 μ l IPTG (200 μ g/ μ l) solution simultaneously.After the incubation, from 1mL bacterium liquid, get 100 μ l and coat on the flat board.37 ℃ of overnight incubation.The evaluation of recombinant clone.

Through cultivating, on the Amp that contains IPTG and X-gal (32g/L) (100g/L) LB flat board, white colony and blue colonies have appearred, and white colony is a recon.

7. the pcr amplification of amplified production and cloned sequence thereof is identified

Utilize the inboard single primer on its joint to increase to amplified production and cloned sequence, use two primer amplifications simultaneously and just can obtain fragment, and list can not increase with a primer, can prove that then amplified fragments and clone are the product of source by homology hybridization in the two DNA samples.

8. dot blot method

Amplified production and cloned sequence are carried out dot hybridization with the amplified production of the micro-separation chromosomal DNA of digoxigenin labeled and total cDNA as probe respectively detect, to determine that further whether amplified production and cloned sequence are from the chromosomal DNA cloning product of micro-separation.

1) some film

The pcr amplification product DNA of clone's of random choose puts the Hybond that grid is arranged drawing one by one ⁺On the nylon membrane (Amersham).With tweezers film is changed in the culture dish, has on the filter paper that soaked into sex change liquid (0.5mol/L NaOH, 1.5mol/L NaCl) bottom it, place 5min.Again film is transferred to and has soaked neutralizer (1.5mol/L NaCl, 0.5mol/L Tris.Cl on filter paper pH7.4), place 8min.Film is transferred on the filter paper that has soaked 2 * SSC, places 5min.Be placed on the dried filter paper room temperature end after the taking-up and do, the DNA irradiation 1 minute that faces down on ultraviolet transilluminator, standby;

2) probe mark: carry out (DIG DNA Labeling and DetectionKit, Boehringer Mannheim) by the description of product

Get the DNA to be marked (chromosomal DNA, cDNA, EcoRI enzyme are cut the rye genomic dna) about 1 μ g, add aseptic double-distilled water to 15 μ l, boil 10min in the boiling water, and put cooled on ice rapidly.In above-mentioned identical Eppendorf pipe, add Hexanucleotide mixed solution 2 μ l, 10 * dNTPs (1mmol/L dATP, 1mmol/L dCTP, 1mmol/L dGTP, 0.65mmol/L dTTP, 0.35mmol/L DIG dUTP, pH7.5) 2 μ l, Klenow enzyme 1 μ l (2U/ μ l), behind the mixing, 37 ℃ of reactions are spent the night.Add 2 μ l 0.2mol/L EDTA (pH8.0) termination reactions.The dehydrated alcohol mixing that adds 2.5 μ l 4mol/LLiCl and 75 μ l precoolings is placed more than the 2h for-20 ℃.The centrifugal 15min of 12000rpm, 70% cold alcohol flushing.Air-dry DNA precipitation, and be dissolved among the 50 μ l TE (pH8.0);

3) prehybridization

In hybridizing box, put into the hybridization nylon membrane, and add an amount of DIG hybridization solution (5 * SSC, 0.1% N-lauroylsarcosine, 0.02%SDS, 10% blocking agent), 65 ℃ are incubated 1h at least;

4) hybridization

In the hybridization solution of 100 μ l, add an amount of probe mixing, sex change 5min in the boiling water, and place on ice rapidly.Remove prehybridization solution, add an amount of hybridization solution and the probe of sex change, shake up (amount that adds hybridization solution will be decided according to the size of film).Seal hybridizing box with adhesive plaster, 65 ℃ of hybridization are spent the night;

5) wash film

Remove hybridization solution, wash 2 times in 2 * SSC, the 0.1% SDS solution under the room temperature, each 5min.Wash twice in 65 ℃, 0.1 * SSC, 0.1% SDS solution, each 15min;

6) detect

(0.1mol/L toxilic acid, 0.15mol/L NaCl pH7.5) embathe 1～5min to Hybond membrane after washing in the toxilic acid damping fluid.Add an amount of (1ml/cm ²) 1 * liquid of blockading (1% blocking agent is dissolved in the toxilic acid damping fluid), reaction 30min.With 1 * blockade liquid dilutedly Gaoxin alkaline phosphatase coupling antibody (1:10000) to 75mU/ml.Film is placed an amount of antibody-solutions room temperature reaction 30min.Wash film 2 times with the toxilic acid damping fluid, each 15min.Film is transferred to (0.1mol/L Tris.Cl, 0.1mol/L NaCl, 50mmol/L MgCl in the detection damping fluid ₂, pH9.5) balance 2～5min.Add an amount of (0.1ml/cm ²) colour developing liquid (NBT and X-phosphate), process color lucifuge and be sure not vibration simultaneously.Colour developing suitably back adds distilled water or the reaction of TE damping fluid color development stopping.Take pictures, write down the result.

Two results

1. technical system determines

Basic line of the present invention is as depicted in figs. 1 and 2: Fig. 2 cuts with the Sau3A enzyme for isolating chromosomal DNA amplified production of micro-dissections and double-stranded cDNA, add that respectively different joints is joint 1 and joint 2, remerge renaturation after the sex change respectively, the result is in last hybridization system outside a spot of strand, will produce two classes hybridization chain, the one, produce by the DNA chain renaturation of same sample source, another kind is the hybridization chain that the homologous fragment renaturation of different sample sources obtains.

Next the fragment of renaturation is mended plain adapter with the Taq enzyme, primer with the joint outside increases, then the strand of renaturation can not be amplified, and the double-stranded both sides that the DNA renaturation in same source produces are with a kind of joint, cause the fragment both sides that long complementary sequence is arranged, in the pcr amplification process, be easy to form so-called panhandle structure (panhandle) and suppressed normal amplified reaction, having only the double-stranded both sides of the DNA renaturation generation of different sources is different joints, can not produce complementary sequence after benefit is flat and also can normally increase, therefore just can selectively amplify the homologous sequence of two DNA samples by this method.

Since in the present embodiment with micro-dissections chromosomal DNA and total cDNA as sample, obtain homologous sequence between micro-dissections chromosomal DNA and total cDNA so can increase by this HSA, Here it is, and we want expressed sequence (Fig. 2) on the isolating karyomit(e).

Utilize strand cDNA and cutting chromosomal DNA hybridization to obtain that the expressed sequence technological line obtains expressed sequence technological line similar (Fig. 1) on the target chromosome with utilizing double-stranded cDNA and the hybridization of cutting chromosomal DNA on the target chromosome, just strand cDNA cuts without enzyme.

2. the pcr amplification of chromosomal micro-separation of rye 1R and DNA thereof

According to preceding method, with the separable acquisition rye of fine glass needle 1R karyomit(e), see Fig. 3 at microscopically.Utilize PCR method can obtain rye 1R chromosomal DNA, see Fig. 4.

3.1R segmental amplification of expressed sequence and clone on the karyomit(e)

Obtained homologous hybridized fragment amplified production among chromosomal DNA and the cDNA by the program in material and the method, the amplified production size of acquisition between 100-500bp (Fig. 5).Amplified fragments T-vector carrier cloning, the evaluation of clone's directly utilizes PN1, PN2 or PN1 and M _4-13Carry out the PCR checking, find to insert fragment between 100-500bp (Fig. 6).

4. identify in amplified production and cloned sequence source

To product and clone's further analysis is that micro-separation 1R chromosomal DNA and total cDNA with digoxigenin labeled carries out dot hybridization as probe to amplified production and cloned sequence, the detected result demonstration does not have the hybridization signal except that unloaded plasmid DNA in contrast, other clones sub-amplified fragments all hybridization signal, show cloned sequence and 1R chromosomal DNA and cDNA homology, further confirmed exactness (Fig. 7 of clone, a, b).

For analyzing clone's the copy number of fragment in genome, rye genomic dna with digoxigenin labeled carries out dot hybridization as probe to cloned sequence, the result shows among 59 clones, have only a clone (6E) that stronger hybridization signal is arranged, this shows that it should be multiple copied sequence (＜2%), other 58 the equal amixia signals of clone are single low segmental clone of copy (〉 98%).This compare with single low copy clone's ratio (about 50%) in the monosome DNA library much higher (Fig. 7, c).

Cloning and sequencing of random choose, sequencing result show that this expressed sequence is thioredoxin gene (Secale cereale thioredoxin-like protein (Trx) mRNA), and this gene is positioned on the 1R karyomit(e) of rye, and sequence is as follows.

1 TTTTTTTTTT?TTCACAAAAC?TAGCATTTTA?CCAGCAGGGT?CATCTTACAG?TTTCACAGCA

61 ATGCATATG?GGCGTCGCAA?AATTGTAAAA?CATTGTCTAG?ACGGAGAAGA?CACGGAAACT

121?GTACACCAGG?AAACTATCAT?GCAACACACA?AAACACACAA?GCATGGCAGC?CAGATATAT

181?AAGCCATGAC?AAGCTGGCTC?TGTTCTAATT?CTGTAGCATG?GTTCAACTGC?CATCACCAAG

241?AGCTTGTACT?TTCTTCTCGA?GCTCAGGTTT?GTTGGCGCCG?ACGAGCTTGT?CGATC

Should be appreciated that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, but the equivalent form of value of changing or revising drops on equally in the application's claims institute restricted portion.

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Claims (7)

1. the method for the chromosomal expressed sequence of separate targets comprises following steps:

(1) separate targets karyomit(e);

(2) acquisition has the target chromosome DNA of joint;

(3) acquisition has total cell cDNA of another kind of joint;

(4) above-mentioned target chromosome DNA and total cell cDNA are carried out homology hybridization;

(5) suppress the pcr amplification homologous sequence, obtain the expressed sequence on the target chromosome,

Wherein obtain target chromosome by the isolating method of little glass needle, and

Wherein the acquisition of target chromosome DNA is to utilize the method for PCR to obtain, and the target chromosome DNA that obtains is cut with the restriction endonuclease enzyme, and add jointing, and the method for described PCR is LA-PCR or DOP-PCR method.

2. according to the process of claim 1 wherein that the acquisition of the cDNA that has another kind of joint is that the total cell mRNA reverse transcription that will extract becomes cDNA, and add top connection.

3. according to the method for claim 1 or 2, wherein the acquisition of expressed sequence is to utilize strand cDNA or double-stranded cDNA on the target chromosome.

4. according to the method for claim 3, wherein the acquisition of expressed sequence comprises that utilizing strand cDNA to carry out target chromosome expressed sequence separates on the target chromosome, and it comprises:

(1) utilizes 3 ' terminal specific primer reverse transcription, first chain, obtain the different cDNA of length;

(2) merge the sex change repeatability with target chromosome DNA, add 100 times of Cot1DNA and blockade, form the hybridization chain of chromosomal DNA and cDNA;

(3) utilize 3 ' the terminal specific primer of joint sequence on the chromosomal DNA and cDNA to suppress pcr amplification hybridization chain, obtain described chromosomal expressed sequence,

Above-mentioned steps (1)-(3) are in the step (3) that is included in the method for claim 1 respectively, (4) and (5).

5. according to the method for claim 3, wherein the acquisition of expressed sequence comprises that utilizing double-stranded cDNA to carry out chromosome expressed sequence separates on the target chromosome, and it comprises:

(1) reverse transcription synthetic double chain cDNA, the restriction endonuclease enzyme is cut, and adds jointing;

(2) DNA with target chromosome or chromosome segment merges the sex change repeatability, adds 100 times of Cot1DNA and blockades, and forms the hybridization chain of chromosomal DNA and cDNA;

(3) utilize the joint sequence of joint sequence on the chromosomal DNA and cDNA to suppress pcr amplification hybridization chain, obtain chromosome expressed sequence,

Above-mentioned steps (1)-(3) are in the step (3) that is included in the method for claim 1 respectively, (4) and (5).

6. according to the method for claim 4 or 5, wherein merging denaturation temperature is 98 ℃, and the reaction times is 1.5 minutes.

7. according to the method for claim 4 or 5, wherein the renaturation temperature is 68 ℃, and the time is 10 hours.

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