CN1782074A - Method for amplifying rice T-DNA insertion site flanking sequence - Google Patents
Method for amplifying rice T-DNA insertion site flanking sequence Download PDFInfo
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Abstract
The present invention discloses a process of amplifying rice The-DNA insertion site flanking sequence. The process includes the following steps: 1. restriction connection through cleaving rice genome DNA with blunt end restrictive endonuclease and connecting the obtained cleaved segment to asymmetrical joint sequence with T4 DNA jointing enzyme in the same reaction system to obtain restriction connecting liquid, with the asymmetrical joint sequence being double stranded joint comprising one long strand and one short strand complementary to each other and having no homogeneous sequence in rice gene and the long strand having at least two nested primer combining sites; and 2. performing twice nested primer PCR amplification with the obtained cleaved segment as template and the nested primer designed according to the asymmetrical joint sequence and T-DNA left boundary sequence to obtain T-DNA insertion site flanking sequence. The present invention is suitable for high flux rice genome analysis.
Description
Technical field
The present invention relates to a kind of method of amplifying rice T-DNA insertion site flanking sequence.
Background technology
Paddy rice is the main food of population over half in the world, is representational cereal crop.Because of its less relatively genome, sophisticated transformation technology system, the good physical map that makes up, two subspecies of a large amount of cDNA sequences and indica and japonica are near the genome project of finishing, and paddy rice has become the modular system of monocotyledons genomics research.
The approach that generally is used for studying gene function is to create DNA to insert mutant colony, utilizes screening mutant and phenotypic evaluation are come sldh gene.It is one of important platform of paddy rice functional genomics research that T-DNA inserts mutant library, and the separation of T-DNA insertion site flanking sequence helps the functional analysis of T-DNA inserted mode and paddy rice Main Agronomic Characters gene.The amplification method of rice T-DNA insertion site flanking sequence has the plasmid rescue method, the terminal rapid amplifying method of cDNA (being divided into 5-end RACE and 3-end RACE method), inverse PCR method (iPCR), hot asymmetric PCR method (TAIL-PCR) etc.Traditional method such as inverse PCR method, its amplification efficiency are subject to the sequence of inserting the site and the restriction enzyme site number of genomic dna, and its sequence fragment size of obtaining of increasing is big inadequately, can not obtain enough homologous sequence information from gene database sometimes.The plasmid rescue method is subjected to the restriction of spawn culture and time very long.TAIL-PCR must carry out multistep PCR under differing temps.These method complex operation step at the bottom of the flanking sequence amplification efficiency, waste time and energy, and are not suitable for the analysis of high-throughput sequence amplification.
At present, the model plant Arabidopis thaliana can be analyzed the plant genome DNA information that T-DNA inserts the fragment side with the PCR-walking method.As shown in Figure 1, its key step is following at first carries out enzyme with the flush end restriction enzyme to plant genome DNA and cuts digestion, then joint is annealed renaturation to the blunt-ended fragment that cuts out.The primer of a pacing (WP) is according to the design of 3 ' side of the left margin sequence (LB) of T-DNA, and another primer is the sequences Design (AP) according to joint.Just can amplify T-DNA with WP and AP and insert the carrier framework sequence that the DNA of plants sequence of fragment flank maybe might be inserted into.Nested WP and the AP primer of general design 2-3 cover carries out the nest-type PRC that 2-3 takes turns, and just can obtain carrying out the PCR product of further institute requirement.But at present, there is not a cover to be suitable for the PCR-walking technical system of paddy rice.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of quick specific amplified rice T-DNA insertion site flanking sequence.
The method of amplifying rice T-DNA insertion site flanking sequence provided by the present invention may further comprise the steps:
1) enzyme is cut connection: in same reaction system, utilize flush end digestion with restriction enzyme oryza sativa genomic dna and utilize T
4Dna ligase is connected the endonuclease bamhi that obtains with asymmetric joint sequence, obtain enzyme and cut connection liquid; Described asymmetric joint sequence is that long-chain and short chain complementation should have higher GC content, and do not have homologous sequence in rice genome by a long-chain and the double-stranded joint that short chain is formed, and long-chain has the binding site of at least two nested primerss;
2) cutting junction fragment with the enzyme in the step 1 is template, carries out two-wheeled nested primers pcr amplification with the nested primers according to the left margin sequences Design of described asymmetric joint sequence and T-DNA, obtains T-DNA and inserts the site flanking sequence.
In order to improve the specificity of amplification, 3 of the short chain of described asymmetric joint sequence ' terminal modified has NH
2Base.
Cut in order to ensure abundant enzyme, the enzyme in the step 1) is cut the ligation system and be can be 10 μ l, and composition and reaction conditions that described enzyme is cut the ligation system are: oryza sativa genomic dna 40-100ng, flush end restriction enzyme 3U, T
4Dna ligase 70U, 1 * T
4The dna ligase damping fluid, asymmetric joint sequence 50 μ M add distilled water to l0 μ l, and 25 ℃ were reacted 6-16 hour.Wherein, agents useful for same is all available from the precious biotechnology in Dalian company limited.
The composition of described reaction system and reaction conditions are preferably oryza sativa genomic dna 82.5ng, flush end restriction enzyme 3U, T
4Dna ligase 70U, T
4Dna ligase damping fluid 1 *, asymmetric joint sequence 50 μ M add ddH
2O 25 ℃, reacted 6-16 hour to 10 μ l.
Described flush end restriction enzyme can be Dra I, EcoR V, Bal I, Pvu II, Sma I, Sna I or Stu I.
Specifically can the serve as reasons single stranded oligonucleotide sequence (ADA1) with sequence 1 in the sequence table and the single stranded oligonucleotide sequence (ADA2) with sequence 2 in the sequence table of described asymmetric joint sequence is heated to 80 ℃, cooling at room temperature then, the double-stranded joint that annealing forms.
Step 2) in the nested primers of two-wheeled described in the pcr amplification, the primer of first round pcr amplification is to can be the single stranded oligonucleotide sequence with sequence 3 in the sequence table and the single stranded oligonucleotide sequence of sequence 4; Second primer of taking turns pcr amplification is to can be the single stranded oligonucleotide sequence with sequence 5 and sequence 6 in the sequence table.
Reclaim and sequencing for the ease of DNA, the reaction system of two-wheeled nested primers pcr amplification all can be 40 μ l.Wherein, the reaction system of described first round pcr amplification can be enzyme and cuts connection liquid 2 μ l, rTaq enzyme 4U, and each 0.3 μ M of forward and reverse primer, dNTP 175 μ M, 1 * PCR damping fluid (can TaKaRa), add distilled water to 40 μ l available from the precious biotechnology in Dalian company limited; Described second reaction system of taking turns pcr amplification can be 50 times of diluents of first round PCR product, 3 μ l, rTaq enzyme 1.5U, and each 0.5 μ M of forward and reverse primer, dNTP 115 μ M, 1 * PCR damping fluid adds distilled water to 40 μ l.
The reaction conditions of described two-wheeled nested primers pcr amplification all can be 94 ℃ of pre-sex change 3min down; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 2.5min down, circulate 35 times; 72 ℃ are fully extended 5min down.
The method of amplifying rice T-DNA insertion site flanking sequence of the present invention has following characteristics: 1) enzyme is cut and is connected in the same reaction system and carries out under the normal temperature, and is fast succinct, saved reaction reagent and reaction times greatly; 2) the asymmetry joint has guaranteed the specificity that increases with nested PCR; The asymmetry joint is because short chain 3 ' end has NH
2Group has prevented that the binding site of joint primer from producing, and the binding site of joint primer only increases under the T-DNA primer; Even amplify the fragment that two ends have joint sequence, also can be owing to inverted terminal repeat sequence forms chiral structure and suppression of amplification when annealing; 3) flush end restriction enzyme (as Dra I or EcoR V) is at T
4In the 10 μ l systems of ligase enzyme damping fluid fully enzyme cut; 4) under the single endonuclease digestion condition, only just can reach amplification efficiency 85%, far above the amplification efficiency of general method with left margin primer and joint primer; 5) for the second time the PCR reaction system reaches 40 μ l and has improved productive rate greatly, is beneficial to DNA and reclaims and sequencing.This method is applicable to high-throughput paddy gene group analysis.
Description of drawings
Fig. 1 is PCR-walking technology amplification T-DNA flanking sequence principle schematic
Fig. 2 catches the physical map of plasmid pFX-E24.2-15R for enhanser
Embodiment
Experimental technique in following examples is ordinary method if no special instructions.
Embodiment 1, paddy rice T1 insert the flanking sequence amplification of mutant for T-DNA
1, paddy rice T1 inserts the acquisition of mutant for T-DNA
It is the enhanser capturing carrier pFX-E24.2-15R (available from Australian CAMBIA research centre) that contains GAL4-VP16-GUS enhanser capture element that rice genetic transforms used T-DNA binary vector.The physical map of carrier has the GAL4-VP16 element that is driven by basic 35S promoter as shown in Figure 2 in the T-DNA right margin.The binding site of GAL4 is six times of UAS on length.The downstream of UAS is a gus gene.The T-DNA district carries required pUC-ori of plasmid rescue and ampicillin resistance gene.The selectable marker gene of vegetable transformant---Totomycin-3 ' NOS---is connected in the place of T-DNA left margin adjacency, is driven by 35S promoter.Selection resistant gene in the bacterium---chloramphenicol resistance gene is in T-DNA left margin (LB) outside.Among Fig. 2, T BORDER (L): T-DNA district left margin; T BORDER (R): T-DNA district right margin; CAM RESISTANCE: chloramphenicol resistance gene; The CAMV35S:35S promotor; HYG (R): hygromycin gene; POLY A SITE:Poly A site; Amp: ampicillin resistance gene; BoGUS: glucuronidase gene; GFP: green fluorescence protein gene; EGFP: enhanced green fluorescence protein gene; 6xUAS:6 section multiple upstream activating sequence; GAL4/VP16: yeast transcription activating protein GAL4DNA is in conjunction with the fusion gene of territory and hsv activation domain.
The super virulent strain of agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105.EHA105 is the bacterium that derives of EHA101, and has deleted T-DNA and the kan resistant gene of the pTiB0542 among the EAH101.With A136 is the karyomit(e) background, amber alkali type (Suc).It is more intense that this bacterial strain has infection ability, and growth is fast, not good characteristic such as balling.
With the callus that pFX-E24.2-15R imports japonica rice variety Japan fine (O.sativa spp.Japonica), obtain paddy rice japonica rice variety Japan fine (O.sativa spp.Japonica) T1 for transformant 325 strains with the gene transformation method of agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 mediation through screening and culturing.
2, amplifying rice T1 inserts the flanking sequence of mutant for T-DNA
Water intaking rice japonica rice variety Japan fine (O.sativa spp.Japonica) T1 extracts oryza sativa genomic dna for the blade of transformant before 325 strain heading stages with the CTAB method, after enzyme is cut connection, as the PCR reaction template.
The double-stranded sequence of joint is respectively:
ADA1:5 '-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCGGGGAGGT-3 ' (sequence 1)
ADA2:5 ' P-ACCTCCCC-NH
23 ' (sequence 2)
ADA1 and ADA2 are heated to 80 ℃, and cooling at room temperature is annealed into double-stranded joint then.
WP1:5 '-CGATGGCTGTGTAGAAGTACTCGC-3 ' (sequence 3)
AP1:5 '-GGATCCTAATACGACTCACTATAGGGC-3 ' (sequence 4)
WP2:5 '-GTTCCTATAGGGTTTCGCTCATGTGTTG-3 ' (sequence 5)
AP2:5 '-CTATAGGGCTCGAGCGGC-3 ' (sequence 6).
WP1 and WP2 are the nested primerss according to the design of T-DNA left margin.AP1 and AP2 are the nested primerss according to joint design.Joint and primer are given birth to worker company by Shanghai and are synthesized.
PCR reagent: rTaq enzyme and dNTP mixture are Dalian precious biotechnology company limited product.
Restriction enzyme: DraI is a Dalian precious biotechnology company limited product.
Ligase enzyme: T
4Dna ligase and damping fluid thereof are available from the precious biotechnology in Dalian company limited.
Molecular weight standard: SD300 is available from ancient cooking vessel state biotechnology limited liability company.
Rice T-DNA insertion site flanking sequence pcr amplification method operation steps:
1) enzyme is cut connection: the enzyme of paddy DNA cut and with the same system of being connected of joint in carry out.The composition of reaction system and reaction conditions are oryza sativa genomic dna 82.5ng, flush end restriction enzyme 3U, T
4Dna ligase 70U, T
4Dna ligase damping fluid 1 * (available from the precious biotechnology in Dalian company limited), asymmetric joint sequence 50 μ M add ddH
2O 25 ℃, reacted 6-16 hour to 10 μ l.
2) first round PCR: reaction system is that the enzyme that step 1) obtains is cut connection liquid 2 μ l, rTaq enzyme 4U, and each 0.3 μ M of forward and reverse primer, dNTP 175 μ M, 1 * PCR damping fluid (available from the precious biotechnology in Dalian company limited) adds distilled water to 40 μ l;
3) second take turns PCR: reaction system is 50 times of diluents of first round PCR product, 3 μ l, rTaq enzyme 1.5U, and each 0.5 μ M of forward and reverse primer, dNTP 115 μ M, 1 * PCR damping fluid adds distilled water to 40 μ l.Reaction conditions is 94 ℃ of pre-sex change 3min down; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 2.5min down, circulate 35 times; 72 ℃ are fully extended 5min down.
4) the PCR product detects: take turns PCR product 2 μ l and sample loading buffer mixing with second, adding contains in the 1.2% sepharose sample hole of 25ng/ml ethidium bromide electrophoresis 20min under 100V voltage.The result shows has 276 sample amplification to go out one or more products, and amplification efficiency is 85%, and each sample strip is counted difference, band position difference.Every band is cut glue, give birth to worker's granulated glass sphere (silver beads) glue with reference to Shanghai and reclaim the recovery of test kit operation steps, detailed step is according to the test kit working instructions.
5) PCR reclaims the dna sequencing of product
Use ABI3730 genetic analysis instrument that the portion of product in the recovery product (80) is checked order according to ordinary method.The result is as shown in table 1, has 7 samples not measure the result, have result that 8 samples measure and carrier sequence and rice genome sequence with BLASTN comparison back without any homology, these two account for 18.7% of order-checking total number of samples.Have the sequencing result of 81.3% PCR-walking product to show the sequence that contains the T-DNA district, wherein 46.2% sequence includes the paddy rice sequence, promptly can tentatively determine the insertion site of conversion elements.
Table 1. part PCR-walking product sequencing result
| Sequencing result | Sample size | Ratio (%) | Enumerate the sample segment strain |
| The successful sequence that checks order contains the T-DNA district, the paddy rice sequence is also arranged, can determine to insert the site and have only the paddy rice sequence, the carrier sequence deletion has only T-DNA region sequence and padding sequence, no paddy rice sequence T-DNA reads over the frontier district, the carrier framework sequence is arranged, and no paddy rice sequence is not measured the result | 65 30 3 14 18 15 | 81.3 46.2 37.5 4.6 3.8 21.5 17.5 27.7 22.5 18.7 | D0261，D0559，D0637， D1670，D1776，D2174-1， D2160，D2174-2，D2174-3， D2816-2，D2816-3，D3757-1 D2104，D0925，D1675-1 D3757-2，D3760，D1797-1， D1797-2，D1944，D1957， D2091，D3775 D1876，D1919，D1921， D2241，D2509，D2688 |
The result of order-checking shows that T-DNA inserts site diversified situation is arranged.The padding sequence that does not have other between T-DNA sequence that has and the paddy rice sequence; The sequence that is filled with the unknown sources that length differs that has; What also have has readed over T-DNA left margin sequence, and the carrier framework sequence (27.7%) outside a large amount of T-DNA districts is arranged; Have only the paddy rice sequence in 3 products, can not find the carrier sequence.
Utilize BLASTN comparison instrument (
Www.ncbi.nlm.nih.gov/blast) carrying out initial analysis to having determined 30 distributions of sequence in rice genome of inserting the site, the result shows that the homologous sequence of the row that check order all has distribution (table 2) except the 7th karyomit(e) in other karyomit(e).Have two to insert fragments as D2174, be inserted into respectively second and the 11 karyomit(e) in.
Table 2. is surveyed and is inserted the distribution of site on each bar karyomit(e) of paddy rice
| Karyomit(e) | Part is inserted sample strain numbering (expected value) |
| Chromosome 1 chromosome 2 chromosomes 3 chromosomes 4 chromosomes 5 chromosomes 6 chromosomes 7 chromosomes 8 chromosomes 9 chromosomes 10 chromosomes 11 chromosomes 12 | D0559(e-130)，D2104(0.11)，D2122(0.01)，D2356(0.0) D2160(2e-31)，D2174-2(e-157) D0328(0.0)，D1432(e-179)，D2270(0.0)，D3350(e-139) D0261(0.0)，D2344-2(e0.21) D2769(e-111)，D3240(0.0) D3617(2e-57) D1395(2e-20)，D1399(e-102)，D1670(6e-48)，D2816-2(e-142) D1776(0.0)，D2899(0.0) D0925(e-104) D1675-1(e-108)，D2174-1(3e-08) D3757-1(0.0) |
The T-DNA flanking sequence amplification of embodiment 2, paddy rice gus gene endosperm specific expression mutant gene group
1, the acquisition of paddy rice gus gene endosperm specific expression mutant
Method with reference to embodiment 1 makes up paddy rice gus gene endosperm specific expression mutant library, get the immature seed of mutant strain in the rice milking stage phase, leaf and root carry out the GUS histochemical stain, filter out paddy rice gus gene endosperm specific expression mutant according to coloration result.
2, the T-DNA flanking sequence of amplifying rice gus gene endosperm specific expression mutant gene group
Joint, primer, PCR reagent, restriction enzyme, ligase enzyme and operation steps are all with embodiment 1.Use ABI3730 genetic analysis instrument that the portion of product in the recovery product (260) is checked order according to ordinary method.The result is as shown in table 3, has 29 samples not measure the result, have result that 2 samples measure and carrier sequence and rice genome sequence with BLASTN comparison back without any homology, these two account for 11.9% of order-checking total number of samples.Have the sequencing result of 88.1% PCR-walking product to show the sequence that contains the T-DNA district, wherein 52.8% sequence includes the paddy rice sequence, promptly can tentatively determine the insertion site of conversion elements.
Table 3. part PCR-walking product sequencing result
| Sequencing result | Sample size | Ratio (%) | Enumerate the sample segment strain |
| The successful sequence that checks order contains the T-DNA district, the paddy rice sequence is also arranged, can determine to insert the site and have only the paddy rice sequence, the carrier sequence deletion has only T-DNA region sequence and padding sequence, no paddy rice sequence | 229 121 6 56 | 88.1 46.5 52.8 2.3 2.6 21.5 24.5 | A248，A260，D124-3，D204-2， E070，E129-1，E129-2，E202-2， E353，E357，E414，E555-1， E608-2，E995，F067-2 G451，M210，N661，S464-2， T063-1，T493 A445-2，E060，E555-2， E555-3，E801，F392-1，F632， |
| T-DNA reads over the frontier district, the carrier framework sequence is arranged, and no paddy rice sequence is not measured the result | 46 31 | 17.7 20.1 11.9 | G402，G548-2，K082-2 D124-1，D204-1，E202-1， E608-1，F206-2，G253-1， G369-1，G465，G548-1，K126 |
Utilize BLASTN comparison instrument (
Www.ncbi.nlm.nih.gov/blast) carrying out initial analysis to having determined 121 distributions of sequence in rice genome of inserting the site, the result shows that the homologous sequence of the row that check order all has distribution (table 4) in each bar karyomit(e).
Table 4. is surveyed and is inserted the distribution of site on each bar karyomit(e) of paddy rice
| Karyomit(e) | Part is inserted sample strain numbering (expected value) |
| Chromosome 1 chromosome 2 chromosomes 3 chromosomes 4 chromosomes 5 chromosomes 6 chromosomes 7 chromosomes 8 chromosomes 9 chromosomes 10 chromosomes 11 chromosomes 12 | G056-2(2e-23)，L351(0.0)，0059(6e-18)，0081-3(2e-23)，0249-1(0.0)，K362(0.0) K082-1(4e-30)，K326-1(9e-46)，M303(1e-22)，N123(5e-48)，P233-2(2e-10) A260(1e-06)，E357(1e-64)，N157-1(0.0)，N157-2(0.0)，0071(3e-25)，T122(0.0) F206-1(3e-49)，K326-2(1e-35)，T150-1(2e-18)，X214(9e-08)，Y655(0.0) P049-2(0.0)，R421(7e-45)，V157(e-158)，Y515(e-154) G369-3(7e-70)，0684(0.0)，R690-1(8e-09)，Z410-1(2e-09) T171(1e-15)，T221(1e-20) L643(4e-16)，M238(2e-13)，Y794(3e-52) E202-2(6e-76)，Q587-2(e-111)，T154(e-154)，N021(4e-66)，R339(0.0) E129-1(6e-24)，E608-2(6e-57)，G369-2(e-134)，V539(e-131)，Y690-3(e-105) E555-1(5e-11)，0094-2(2e-09)，P357(1e-14)，T063-1(e-178)，Y438(e-112) F542(9e-33)，W663-1(4e-30)，L172-1(0.0) |
Sequence table
<160>6
<210>1
<211>8
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>1
acctcccc 8
<210>2
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>2
cgatggctgt gtagaagtac tcgc 24
<210>3
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>3
ggatcctaat acgactcact atagggc 27
<210>4
<211>43
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>4
ctaatacgac tcactatagg gctcgagcgg ccgccgggga ggt 43
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>5
gttcctatag ggtttcgctc atgtgttg 28
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>6
ctatagggct cgagcggc 18
Claims (9)
1, a kind of method of amplifying rice T-DNA insertion site flanking sequence may further comprise the steps:
1) enzyme is cut connection: in same reaction system, utilize flush end digestion with restriction enzyme oryza sativa genomic dna and utilize T
4Dna ligase is connected the endonuclease bamhi that obtains with asymmetric joint sequence, obtain enzyme and cut connection liquid; Described asymmetric joint sequence is by a long-chain and the double-stranded joint that short chain is formed, long-chain and short chain complementation, and in rice genome, do not have homologous sequence, long-chain has the binding site of at least two nested primerss;
2) cutting junction fragment with the enzyme in the step 1 is template, carries out two-wheeled nested primers pcr amplification with the nested primers according to the left margin sequences Design of described asymmetric joint sequence and T-DNA, obtains T-DNA and inserts the site flanking sequence.
2, method according to claim 1 is characterized in that: 3 of the short chain of the described asymmetric joint sequence ' terminal modified NH of having
2Base.
3, method according to claim 2 is characterized in that: composition and reaction conditions that described enzyme is cut the ligation system are: oryza sativa genomic dna 40-100ng, flush end restriction enzyme 3U, T
4Dna ligase 70U, T
4The dna ligase damping fluid, asymmetric joint sequence 50 μ M add distilled water to 10 μ l, and 25 ℃ were reacted 6-16 hour.
4, method according to claim 3 is characterized in that: the composition of described reaction system and reaction conditions are oryza sativa genomic dna 82.5ng, flush end restriction enzyme 3U, T
4Dna ligase 70U, T
4Dna ligase damping fluid 1 *, asymmetric joint sequence 50 μ M add ddH
2O 25 ℃, reacted 6-16 hour to 10 μ l.
5, according to claim 1,2,3 or 4 described methods, it is characterized in that: described flush end restriction enzyme is Dra I, EcoRV, Bal I, Pvu II, Sma I, Sna I or Stu I.
6, according to claim 1,2,3 or 4 described methods, it is characterized in that: described asymmetric joint sequence is to be heated to 80 ℃ by single stranded oligonucleotide sequence with sequence 1 in the sequence table and the single stranded oligonucleotide sequence with sequence 2 in the sequence table, cooling at room temperature then, the double-stranded joint that annealing forms.
7, method according to claim 6 is characterized in that: in the described two-wheeled nested primers pcr amplification, the primer of first round pcr amplification is to for having the single stranded oligonucleotide sequence of sequence 3 and sequence 4 in the sequence table; Second primer of taking turns pcr amplification is to for having the single stranded oligonucleotide sequence of sequence 5 and sequence 6 in the sequence table.
8, method according to claim 7 is characterized in that: the reaction system of described first round pcr amplification is that enzyme is cut connection liquid 2 μ l, rTaq enzyme 4U, and each 0.3 μ M of forward and reverse primer, dNTP 175 μ M, 1 * PCR damping fluid adds distilled water to 40 μ l; Described second reaction system of taking turns pcr amplification is 50 times of diluents of first round PCR product, 3 μ l, rTaq enzyme 1.5U, and each 0.5 μ M of forward and reverse primer, dNTP115 μ M, 1 * PCR damping fluid adds distilled water to 40 μ l.
9, method according to claim 8 is characterized in that: the reaction conditions of described two-wheeled nested primers pcr amplification is 94 ℃ of pre-sex change 3min down; 94 ℃ of following sex change 30s then, 67 ℃ of annealing 45s down, 72 ℃ are extended 2.5min down, circulate 35 times; 72 ℃ are fully extended 5min down.
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