CN100513577C - PCR method - Google Patents
PCR method Download PDFInfo
- Publication number
- CN100513577C CN100513577C CNB200510080550XA CN200510080550A CN100513577C CN 100513577 C CN100513577 C CN 100513577C CN B200510080550X A CNB200510080550X A CN B200510080550XA CN 200510080550 A CN200510080550 A CN 200510080550A CN 100513577 C CN100513577 C CN 100513577C
- Authority
- CN
- China
- Prior art keywords
- sequence
- reaction
- sitefinder
- amplified
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 36
- 101100448444 Caenorhabditis elegans gsp-3 gene Proteins 0.000 claims abstract description 23
- 101100393821 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GSP2 gene Proteins 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims description 47
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 108091008146 restriction endonucleases Proteins 0.000 claims description 24
- 229940046166 oligodeoxynucleotide Drugs 0.000 claims description 18
- 102100022887 GTP-binding nuclear protein Ran Human genes 0.000 claims description 15
- 101000774835 Heteractis crispa PI-stichotoxin-Hcr2o Proteins 0.000 claims description 15
- 101000620756 Homo sapiens GTP-binding nuclear protein Ran Proteins 0.000 claims description 15
- 238000000137 annealing Methods 0.000 claims description 11
- 101000805921 Strongylocentrotus purpuratus Upstream stimulatory factor Proteins 0.000 claims description 10
- 101000671634 Xenopus borealis Upstream stimulatory factor 1 Proteins 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 6
- 108700005078 Synthetic Genes Proteins 0.000 claims description 4
- 230000004087 circulation Effects 0.000 claims description 4
- 238000010792 warming Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 20
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 102000053602 DNA Human genes 0.000 abstract description 6
- 210000000349 chromosome Anatomy 0.000 abstract description 4
- 238000010367 cloning Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract 1
- 101100171666 Arabidopsis thaliana SFP2 gene Proteins 0.000 description 16
- 101100422767 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SUL1 gene Proteins 0.000 description 16
- 230000003321 amplification Effects 0.000 description 13
- 238000003199 nucleic acid amplification method Methods 0.000 description 13
- 238000007852 inverse PCR Methods 0.000 description 9
- 230000009182 swimming Effects 0.000 description 9
- 101150003216 SFP1 gene Proteins 0.000 description 8
- 238000012216 screening Methods 0.000 description 6
- 241000219194 Arabidopsis Species 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 241000219195 Arabidopsis thaliana Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 101100074337 Drosophila melanogaster lectin-37Da gene Proteins 0.000 description 1
- 101100074338 Drosophila melanogaster lectin-37Db gene Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000007854 ligation-mediated PCR Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 230000000979 retarding effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a PCR method that has high specificity and is easy to operate. It includes the following methods: inducing SiteFinder sequence into gene sequence, taking two circles of PCR index expanding for target DNA molecule; selectively cloning target molecule through limited inscribe enzyme recognition site; selecting target molecule by the GSP3 specificity of the GSP2 lower reaches on known sequence. The invention has the advantages of easy, fast, high specificity, strong commonality, good repeatability, etc. It could be widely used for kinds of target chromosome walking in molecule biology research.
Description
Technical field
The present invention relates to a kind of PCR method.
Background technology
Look into the more traditional establishment gene bank and selection of sequence that adjoins that method obtains to insert the site by PCR step and compare, easy and simple to handle, quick and with low cost, be to separate the unknown nucleotide sequence that adjoins with known array or the first-selected experimental technique of new gene.The existing P CR step is looked into method, as inverse PCR, ligation-mediated PCR, TAIL-PCR etc. exist that method is too complicated, drawbacks such as the fragment little (not reaching the desired length of investigator) of the restriction that is subjected to restriction enzyme site, amplification, poor specificity, usually increase less than the needed dna fragmentation of investigator.
The PCR method that is used to separate the unknown nucleotide sequence that adjoins with known array has at present been reported tens kinds, by its principle that adopts, can reduce three kinds:
1, inverse PCR (Inverse PCR): unknown nucleotide sequence end reflexed is returned, be connected, make PCR with two primers on the known array again with known array.Inverse PCR mainly contains two technical problems: (1) requires with the unknown nucleotide sequence end identical restriction enzyme site to be arranged at known array, and when an end does not have or two ends when all not having proper restriction site, inverse PCR is inapplicable.(2) when all there is proper restriction site in two ends, inverse PCR is very ineffective yet, and is intermolecular because the ligation pilosity is born in, and the probability that intramolecularly connects (cyclisation) is very little.
2, the PCR that connects mediation.The PCR that connects mediation is joint on the unknown nucleotide sequence end connects, and makes pcr amplification with the special primer of known array end and the joint primer of unknown nucleotide sequence end.The common feature of this class PCR method is a poor specificity.
3, annealed PCR (randomly primed PCR) at random: this class PCR also has two technical problems, 1) this class PCR requires to have at the unknown nucleotide sequence end binding site of degenerated primer (arbitrary degenarated primer), and practical situation are the binding sites that lack degenerated primer under many circumstances at the unknown nucleotide sequence end.2) the non-specific amplification problem is very outstanding, and non-specific amplification has been covered specific amplification as a rule, thereby can not get the band of needed specific PCR amplified production.
Summary of the invention
The purpose of this invention is to provide and a kind ofly have than high specific, and PCR method simple to operate.
PCR method provided by the present invention is called SiteFind-PCR, may further comprise the steps:
1) with the initial PCR reaction of SiteFinder;
Described SiteFinder comprises 4
6Plant strand oligodeoxynucleotide molecule, the length of described various strand oligodeoxynucleotide molecules is identical, is 50-100nt; The sequence that 4 to 6 deoxynucleotides of 3 of described various strand oligodeoxynucleotide molecules ' end are formed is all identical, it is all different closely to be connected in the sequence that 6 deoxynucleotides of 5 ' direction of these 4 to 6 deoxynucleotides form, and the sequence of 38-90 deoxynucleotide composition of 5 ' direction that closely is connected in these 6 deoxynucleotides is all identical;
2) carry out the nest-type PRC reaction;
The primer of described nest-type PRC reaction is by according to SiteFinder synthetic SPF1 and SPF2 with according to the known array synthetic gene specific primer GSP1 adjacent with target sequence to be amplified, and GSP2 and GSP3 form; The sequence of described SPF1 and SPF2 all is selected from the sequence that 38-90 deoxynucleotide of described various strand oligodeoxynucleotide molecules of the SiteFinder of step 1) formed; 3 ' end sequence of described SPF1 is identical with 5 ' end sequence of SPF2, forms nested primers;
Described GSP1, among GSP2 and the GSP3, secondly the most approaching target sequence to be amplified of described GSP3 is GSP2, from target sequence to be amplified farthest be GSP1; The binding site of first deoxynucleotide of 3 of described GSP3 ' end and described known array, and 5nt at least apart between last deoxynucleotide of 3 ' end of described known array.
Wherein, for the ease of the PCR product of target sequence to be amplified being selected and the PCR product cloning that obtains being gone into carrier, in the sequence of described 38-90 deoxynucleotide composition, the sequence that closely is connected in the deoxynucleotide composition of described 6 deoxynucleotide 5 ' directions is the single stranded sequence of 5 of a restriction endonuclease recognition sequence ' → 3 ' trend; The recognition sequence of described restriction endonuclease is more than or equal to eight nucleotide pairs.Wherein, the recognition sequence of any restriction endonuclease of forming more than or equal to eight nucleotide pairs all can be realized this purpose, and the reason of using the rare recognition sequence of the restriction endonuclease of forming more than or equal to eight nucleotide pairs is to avoid the target sequence beyond the SiteFinder is cut off.
Also comprise the restriction endonuclease of utilizing the restriction endonuclease recognition sequence correspondence in the various strand oligodeoxynucleotide molecules that described SiteFinder comprises in the described method, the step that the PCR product of target sequence to be amplified is selected.
Recognition sequence comprises NotI (GCGGCCGC) more than or equal to the restriction endonuclease of eight nucleotide pairs, ASC (GGCGCGCC), AsiSI (GCGATCGC), FseI (GGCCGGCC) etc.
The sequence of described SPF1 and SPF2 all is selected from the described 40-90 deoxynucleotide, closely is connected in the sequence that the deoxynucleotide of 5 ' direction of described restriction endonuclease recognition sequence is formed.
Described initial PCR reaction comprises that strand oligodeoxynucleotide molecule among template sex change, the SiteFinder and template annealing, folk prescription are to extension;
The strand oligodeoxynucleotide molecule among the described SiteFinder and the temperature condition of template annealing can be 16-25 ℃ 1 minute; The temperature condition of the template sex change of described initial PCR reaction can be earlier 92 ℃ 2 minutes, then 95 ℃ 1 minute.
The folk prescription of described initial PCR reaction to the temperature condition that extends at least 3 minutes, to be warming up to 68 ℃, then 68 ℃ 10 minutes.
The reaction of described nest-type PRC comprises that the first round and second takes turns amplified reaction, the reaction that a pair of primer that described first round amplified reaction is made up of SPF1 and GSP1 carries out, and described second takes turns the reaction that amplified reaction comprises that a pair of primer be made up of SPF2 and GSP2 carries out.
For according to SPF2 and GSP
2, GSP
3The difference in size of institute's amplification of DNA fragments recognizes whether the PCR product is specific molecular, and described second takes turns the reaction that amplified reaction comprises that also a pair of primer be made up of SPF2 and GSP3 carries out.
The described first round amplified reaction and second temperature condition of taking turns amplified reaction be earlier 94 ℃ 1 minute; 95 ℃ of 10-15 seconds then, 68 ℃ 6 minutes, carry out 30-35 circulation; Last 72 ℃ 5 minutes.
The present invention at first unique design be used for initial PCR reaction by 4
6Plant the molecular SiteFinder of strand oligodeoxynucleotide.Fig. 1 shows a kind of SiteFinder, and its 3 ' end has 4 special Nucleotide, be used for dna profiling on corresponding site complementary pairing, there are 6 random nucleotides these 4 special Nucleotide upstreams, are used for auxiliary 3 ' terminal annealing; And then by the single stranded sequence of 5 ' → 3 ' trend of the recognition sequence of a NotI who forms by 8 Nucleotide, be used for optionally cloning target molecule.
5 ' terminal sequence of the various strand oligodeoxynucleotide molecular sequences that comprise based on SiteFinder, a Synthetic 2 nested primers, i.e. SFP
1And SFP
2Fig. 1 shows the 5 ' terminal sequence synthetic SFP according to above-mentioned SiteFinder
1And SFP
2
According to the known array adjacent with target sequence to be amplified, synthetic gene special primer GSP1, GSP2 and GSP3.GSP1, among GSP2 and the GSP3, secondly the most approaching target sequence to be amplified of described GSP3 is GSP2, from target sequence to be amplified farthest be GSP1.Whether the product that obtains that increases for the ease of identifying is aim sequence, the binding site of first deoxynucleotide of 3 of described GSP3 ' end and described known array, and 5nt at least apart between last deoxynucleotide of 3 ' end of described known array.
Shown in the principle of SiteFind-PCR such as Fig. 2 (thick line shows known array, and fine rule is a unknown nucleotide sequence):
(1) genomic dna; (2) by template sex change, SiteFinder at low temperatures with genomic templates annealing, folk prescription to extension, the SiteFinder sequence is introduced among the genome sequence; (3) by the nest-type PRC index target dna molecule that doubly increases, but not target molecule can't be finished the index amplification because of the restraining effect of loop-stem structure, thereby reaches the purpose of selective amplification target molecule; (4) optionally clone target molecule by restriction endonuclease recognition sequence (as the NotI recognition sequence); (5) GSP3 by GSP2 downstream on the known array screens target molecule specifically.
In general, in order to guarantee the specificity of PCR product, in the PCR process, should avoid non-special initial generation as far as possible.But, this non-special initial with respect to whole primer.For several bases of 3 ' of primer-end, even wrong initial, also must be accurately with template DNA molecule paired, otherwise can't finish the amplification of dna molecular.Utilize this characteristics, rely on 3 ' of SiteFinder-4-6 terminal base to seek on the genome and its paired site, the amplification (Fig. 2) of starting the dna molecular in downstream, corresponding site on the whole genome.Finished selection subsequently: select for the first time (selective amplification) to target molecule, target molecule is under the effect of gene specific primer, the complementary strand of SiteFinder is filled complete, thereby obtain having only an end that the SiteFinder sequence is arranged and the other end is the dna molecular of known array.This molecule can doubly be increased by index in the PCR process.Yet for non-target dna molecule, most molecules will only can have the SiteFinder sequence at an end of molecule, and they only can be by linear amplification in subsequent P CR process; Even there are a few molecules two ends all to have the SiteFinder sequence, because they constitute inverted repeats at the two ends of molecule, to form stable loop-stem structure because space length is approaching in the sex change annealing process of PCR, this structure has stoped the annealing of primer and template.Select for the second time (selectivity clone), on SiteFinder, designed the recognition sequence (as the NotI recognition sequence) of a restriction enzyme, greatly facilitate clone on the one hand, the more important thing is and again target molecule is selected target molecule.Having only target molecule just to satisfy an end in theory is the recognition sequence (as the NotI recognition sequence) of this restriction enzyme, an other end is a flush end, thereby is connected to in this restriction enzyme (as NotI) and the linearizing carrier of another endonuclease (cutting as the EcoRV enzyme) (as pBlueScript SK (+) plasmid).Non-target molecule has the recognition sequence (as the NotI recognition sequence) of this restriction enzyme because of two ends or recognition sequence (as the NotI recognition sequence) the other end that an end is this restriction enzyme is ring texture (loop), and can not be cloned.Select for the third time (selective screening), right product rather than the right product of GSP3/SFP2 primer of clone's GSP2/SFP2 primer, when purpose is to guarantee to use carrier primer and GSP3 screening, have only the product of the specific amplified of target molecule can screened arriving, and the molecule in other sources all can not be screened come out.Have only the right amplified production length of GSP3/SFP2 primer less than the right amplified production of GSP2/SFP2 primer, be only the product of the specific amplified of target molecule.
SiteFind-PCR of the present invention has the following advantages:
1. simple to operate: as not have in the operating process that enzyme is cut, precipitated, recovery and a connection procedure, simplified operation steps greatly, reduced contaminated probability.
2. high specific: look in the existing step and to have only inverse PCR in the PCR method (Inverse PCR) and T-linker PCR have high specific (Yan, Y.X., An, C.C., Li, L., Gu, J.Y., Tan, G.H., and Chen, Z.L. (2003) .T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of taggedDNA ends.Nucleic Acids Res.30, e68.
), but they all are subjected to the restriction of restriction enzyme site, and for inverse PCR, and the target dna molecule cyclisation difficulty under nonselective condition of contact in the genome, causing is having under the situation of suitable restriction enzyme site also the difficulty target dna molecule that increases.Other step is looked into the PCR method poor specificity, increase often with the incoherent non-specific molecules of research purpose.Because the retarding effect of loop-stem structure among the SiteFind-PCR, and follow-up selectivity is cloned and screening, has guaranteed result's high specific.
3. high success rate: in the experiment of being carried out, successfully from 8 samples, obtain 8 positive findingses, illustrate for each sample and can both be looked into by effective stage.
4. obtain big segment easily: because without the restriction enzyme processing template, its initiation site only is subjected to the restriction of the amplification ability of Taq enzyme to the ultimate range of known array in theory, is easy to obtain bigger segment so do chromosome walking with SiteFind-PCR.
SiteFind-PCR compares with existing PCR method, has advantages such as simple to operate, quick, that specificity is high, highly versatile, good reproducibility, amplified fragments are long and with low cost.SiteFind-PCR can be widely used in the chromosome walking of multiple purpose in the molecular biology research, inserts the evaluation in site, the separation of the new gene of the unknown section of gene etc. as foreign gene in transgenosis animal and plant and the microorganism.
Description of drawings
Fig. 1 is a SiteFinder synoptic diagram and according to the sequence of its synthetic SFP1 and SFP2
Fig. 2 is the schematic diagram of SiteFind-PCR
Fig. 3 is the sequence of DL3, DL2 and DL1
Fig. 4 for identify with SiteFind-PCR first Arabidopis thaliana insert mutant the insertion site second take turns PCR product electrophoretogram and to the sequencing result of clone's product
Fig. 5 for identify simultaneously with SiteFind-PCR 7 Arabidopis thalianas insert mutant the insertion site second take turns PCR product electrophoretogram
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
One, the acquisition of arabidopsis mutant body
With pSki015 (WeigelWorld) is the carrier of arabidopsis thaliana transformation, according to document (Chang, S.S., Park, S.K., Kim, B.C., Kang, b.J., Kim, D.U., and Nam, H.G. (1994) .Stable genetic transformation of Arabidopsis thaliana byAgrobacterium inoculation in planta.Plant J.5,551-558.; Gelvin, S.B. (2003) .Agrobacterium-mediated plant transformation:thebiology behind the " gene-jockeying " tool.Microbiol Mol Biol Rev 67,16-37, table of contents; Tzfira, T., Li, J., Lacroix, B., andCitovsky, V. (2004) .Agrobacterium T-DNA integration:molecules andmodels.Trends Genet 20, method 375-383), with flower-dipping method arabidopsis thaliana transformation (Colombia type ecospecies), through herbicide screening, the T-DNA that obtains 8 strain antiweeds in the plant of first filial generation inserts mutant.
Two, identify that with SiteFind-PCR 1 Arabidopis thaliana inserts the insertion site of mutant
1, SiteFinder, SFP1 and SFP2, GSP3, the design of GSP2 and GSP1 is synthetic
(1) the synthetic SiteFinder of design
Designed synthetic SiteFinder comprises 4
6Plant strand oligodeoxynucleotide molecule SiteFinder, SiteFinder-4096.Their size is 61nt, their sequence general formula: 5 '-CACGACACGCTACTCAACACACCACCTCGCACAGCGTCCTCAAGCGGCCGCNNNNN NGCCT-3 ', wherein, 3 ' 4 terminal special Nucleotide GCCT be used for dna profiling on corresponding site complementary pairing, there are 6 random nucleotide NNNNNN (N is A or T or C or G) these 4 special Nucleotide upstreams, are used for assisting 3 ' terminal annealing; And then by the single stranded sequence GCGGCCGC of recognition sequence 5 ' → 3 ' trend of a NotI who forms by 8 Nucleotide, be used for optionally cloning target molecule.
(2) synthetic SFP1 of design and SFP2
5 ' terminal sequence of the various strand oligodeoxynucleotide molecular sequences that comprise based on SiteFinder, Synthetic 2 nested primers SFP1 and SFP2.SFP1?5’-CACGACACGCTACTCAACAC-3’，SFP25’-ACTCAACACACCACCTCGCACAGC-3’。
(3) the synthetic GSP3 of design, GSP2 and GSP1: with near the nucleotide sequence the left margin sequence of shuttle vectors pSki015 as known array, synthetic gene special primer GSP3 (DL3), GSP2 (DL2) and GSP1 (DL1).The sequence of DL3 is 5 '-GCTTTCGCCTATAAATACGACGG-3 ', and the sequence of DL2 is 5 '-TGGACGTGAATGTAGACACGTCGA-3 ', the sequence of DL1 be 5 '-GACAACATGTCGAGGCTCAGCAGG-3 ' (Fig. 3).The initial binding site of DL3 and sequence 1 be sequence 1 from 5 ' end 172-194 position deoxynucleotide, the initial binding site of DL2 and sequence 1 be sequence 1 from 5 ' end 113-136 position deoxynucleotide, the initial binding site of DL1 and sequence 1 be sequence 1 from 5 ' hold 1-24 position deoxynucleotide.
2, with the initial PCR reaction of SiteFinder
The genomic dna of a strain arabidopsis mutant body that obtains with step 1 is a template, with the initial PCR reaction of SiteFinder.Wherein, reaction system is 20 μ L, comprises 0.1 μ g genomic dna (1 μ L), the SiteFinder of 1 μ L5 μ mol/L, the Taq enzyme of 1 unit, the dNTP of 1 μ L2.5mM.The temperature condition of reaction is 92 ℃ (2min) of elder generation, and 95 ℃ (1min) make the template sex change then; 25 ℃ (1min) make SiteFinder and template annealing again; In 3 minutes, be warming up to 68 ℃, then at 68 ℃ (10min), make SiteFinder to 3 ' direction folk prescription to extension.
3, with SFP1 and SFP2, DL3, DL2 and DL1 carry out the nest-type PRC reaction
In 20 μ L reaction systems of the initial PCR of completing steps 2 reaction, add the SFP1 of 3.5 μ L, 10 μ mol/L, the DL1 of 1 μ L, 10 μ mol/L, 0.5 μ L, 10 * Taq enzyme buffer liquid carries out first round amplified reaction, temperature condition be first 94 ℃ 1 minute; Then 95 ℃ 10 seconds, 68 ℃ 6 minutes, carry out 30 circulations; Last 72 ℃ 5 minutes.
After first round amplified reaction finishes, get 1000 times of 1 μ L product dilutions, get 1 μ L cut back again and make template, the SFP2 that in reaction system, adds 2 μ L, 10 μ mol/L again, the DL2 of 2 μ L, 10 μ mol/L, the dNTP of 0.5 μ L 10mM, the Taq enzyme of 1 unit, water is supplied 50 μ L, carries out second and takes turns amplified reaction.Establish parallel control by above-mentioned system simultaneously, different is to replace DL2 with DL3.Temperature condition be earlier 94 ℃ 1 minute; Then 95 ℃ 10 seconds, 68 ℃ 6 minutes, carry out 30 circulations; Last 72 ℃ 5 minutes.
Second takes turns after amplified reaction finishes, the PCR product that obtains is carried out electrophoresis, electrophoresis result is shown in Fig. 4 A, swimming lane 1 and 2 is respectively SFP2/DL2 and the product of SFP2/DL3 primer to increasing, obtain 3 bands in the swimming lane 1, obtain 2 bands in the swimming lane 2, two bigger in the swimming lane 1 bands are bigger than corresponding two bands in the swimming lane 2 respectively, illustrate that the PCR product that obtains is the specific amplified product of target molecule.
When using carrier primer M13R and DL3 screening positive clone, have only the product of the specific amplified of target molecule can screened arriving, and the molecule in other sources all can not be screened come out, the product that the DL2/SFP2 primer is right is cloned.Reclaim the corresponding dna fragmentation of band (2.2kb) maximum in the swimming lane 1, after cutting with the NotI enzyme, with be connected with the carrier pBlueScript SK (+) that the EcoRV enzyme is cut through NotI, to connect the product transformed into escherichia coli according to a conventional method, carry out bacterium colony PCR screening positive clone with M13R and DL3, check order, sequencing result shows that the length of the dna fragmentation of band correspondence maximum in the swimming lane 1 is 2270bp, in this fragment, a GCCT site (Fig. 4 B) is arranged, this and electrophoresis result match (band of white arrow indication among Fig. 4 A) at the 783bp place of distance DL3.Sequencing result shows that obtaining band maximum in the swimming lane 1 is 2.2kb.
Sequence alignment among resulting sequence and the GeneBank is analyzed T-DNA insert the site.The result shows that in this arabidopsis mutant body the segmental on position of external source is on No. three karyomit(e) of arabidopsis gene group.This qualification result that inserts the site is identical with the insertion site of pressing the TAIL-PCR evaluation, but the segment that the step looks into is than the long 1.6kb of segment that looks into by the TAIL-PCR step.
In order to verify the extensive applicability of SiteFind-PCR, other 7 mutant in embodiment 1 step 1 are inserted the analysis in site.
Removing template is respectively outside the genomic dna difference of these 7 mutant, and other reaction material (as SiteFinder, GSP3, GSP2 and GSP1, SFP1 is SFP2) all identical with embodiment 1 with reaction conditions.The result judges have 7 samples to obtain the specific amplified product according to the difference of electrophoretic band as shown in Figure 5, and wherein sample 4 has the product of 3 specific amplifieds.Among Fig. 5, swimming lane II and III are the SFP2/DL2 and the right amplified production of SFP2/DL3 primer of each mutant sample; 1-7 represents the sample of 7 mutant respectively.
Sequence table
<160>
<210>1
<211>421
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
Claims (10)
1, a kind of PCR method may further comprise the steps:
1) with the initial PCR reaction of SiteFinder;
Described SiteFinder comprises 4
6Plant strand oligodeoxynucleotide molecule, the length of described various strand oligodeoxynucleotide molecules is identical, is 50-100nt; The sequence that 4 to 6 deoxynucleotides of 3 of described various strand oligodeoxynucleotide molecules ' end are formed is all identical, it is all different closely to be connected in the sequence that 6 deoxynucleotides of 5 ' direction of these 4 to 6 deoxynucleotides form, and the sequence of 38-90 deoxynucleotide composition of 5 ' direction that closely is connected in these 6 deoxynucleotides is all identical;
2) carry out the nest-type PRC reaction;
The primer of described nest-type PRC reaction is by according to SiteFinder synthetic SPF1 and SPF2 with according to the known array synthetic gene specific primer GSP1 adjacent with target sequence to be amplified, and GSP2 and GSP3 form; The sequence of SPF1 and SPF2 all is selected from the sequence that 38-90 deoxynucleotide of described various strand oligodeoxynucleotide molecules of the SiteFinder of step 1) formed; 3 ' end sequence of described SPF1 is identical with 5 ' end sequence of SPF2, forms nested primers;
Described GSP1, among GSP2 and the GSP3, secondly the most approaching target sequence to be amplified of described GSP3 is GSP2, from target sequence to be amplified farthest be GSP1; The binding site of first deoxynucleotide of 3 of described GSP3 ' end and described known array, and 5nt at least apart between last deoxynucleotide of 3 ' end of described known array.
2, method according to claim 1, it is characterized in that: in the sequence of described 38-90 deoxynucleotide composition, the sequence that closely is connected in the deoxynucleotide composition of described six deoxynucleotide 5 ' directions is the single stranded sequence of 5 of a restriction endonuclease recognition sequence ' → 3 ' trend; The recognition sequence of described restriction endonuclease is more than or equal to eight nucleotide pairs.
3, method according to claim 2, it is characterized in that: also comprise the restriction endonuclease of utilizing the restriction endonuclease recognition sequence correspondence in the various strand oligodeoxynucleotide molecules that described SiteFinder comprises in the described method, the step that the PCR product of target sequence to be amplified is selected.
4, method according to claim 2, it is characterized in that: the sequence of described SPF1 and SPF2 all is selected from the described 38-90 deoxynucleotide, closely is connected in the sequence that the deoxynucleotide of 5 ' direction of described restriction endonuclease recognition sequence is formed.
5, according to claim 1,2,3 or 4 described methods, it is characterized in that: described initial PCR reaction comprises that strand oligodeoxynucleotide molecule among template sex change, the SiteFinder and template annealing, folk prescription are to extension; The strand oligodeoxynucleotide molecule among the described SiteFinder and the temperature condition of template annealing be 16-25 ℃ 1 minute.
6, method according to claim 5 is characterized in that: the temperature condition of described template annealing is 25 ℃ of 1min; The temperature condition of the template sex change of described initial PCR reaction be first 92 ℃ 2 minutes, then 95 ℃ 1 minute.
7, method according to claim 5 is characterized in that: the folk prescription of described initial PCR reaction to the temperature condition that extends at least 3 minutes, to be warming up to 68 ℃, then 68 ℃ 10 minutes.
8, according to claim 1,2,3 or 4 described methods, it is characterized in that: the reaction of described nest-type PRC comprises that the first round and second takes turns amplified reaction, the reaction that a pair of primer that described first round amplified reaction is made up of SPF1 and GSP1 carries out, described second takes turns the reaction that amplified reaction comprises that a pair of primer be made up of SPF2 and GSP2 carries out.
9, method according to claim 8 is characterized in that: described second takes turns the reaction that amplified reaction comprises that also a pair of primer be made up of SPF2 and GSP3 carries out.
10, method according to claim 8 is characterized in that: the described first round amplified reaction and second temperature condition of taking turns amplified reaction be earlier 94 ℃ 1 minute; 95 ℃ of 10-15 seconds then, 68 ℃ 6 minutes, carry out 30-35 circulation; Last 72 ℃ 5 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510080550XA CN100513577C (en) | 2005-07-04 | 2005-07-04 | PCR method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510080550XA CN100513577C (en) | 2005-07-04 | 2005-07-04 | PCR method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1891832A CN1891832A (en) | 2007-01-10 |
| CN100513577C true CN100513577C (en) | 2009-07-15 |
Family
ID=37597026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB200510080550XA Expired - Fee Related CN100513577C (en) | 2005-07-04 | 2005-07-04 | PCR method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100513577C (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012070618A1 (en) | 2010-11-24 | 2012-05-31 | 株式会社カネカ | Amplified nucleic acid detection method and detection device |
| JP6219816B2 (en) | 2012-04-27 | 2017-10-25 | 株式会社カネカ | Nucleic acid amplification method and amplified nucleic acid detection method |
| JP6691380B2 (en) | 2013-11-22 | 2020-04-28 | 株式会社カネカ | Method for detecting short RNA |
| CN107385065B (en) * | 2017-08-16 | 2020-07-28 | 北京东方亚美基因科技研究院有限公司 | Method for amplifying nucleic acid and use thereof |
-
2005
- 2005-07-04 CN CNB200510080550XA patent/CN100513577C/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Q-SiteFinder :an energy-based method for the prediction ofprotein-ligand binding sites. Alasdair T.R.Laurie et al.BIOINFORMATICS,Vol.21 No.9. 2005 |
| Q-SiteFinder :an energy-based method for the prediction ofprotein-ligand binding sites. Alasdair T.R.Laurie et al.BIOINFORMATICS,Vol.21 No.9. 2005 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1891832A (en) | 2007-01-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bashyam et al. | A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements | |
| Erauso et al. | Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile | |
| Hochhut et al. | CTnscr94, a conjugative transposon found in enterobacteria | |
| JPWO2019236566A5 (en) | ||
| CN102597257A (en) | Specific method for preparing joined DNA fragments including sequences derived from target genes | |
| CN100513577C (en) | PCR method | |
| CN110607320A (en) | Plant genome directed base editing framework vector and application thereof | |
| CN113717960B (en) | Novel Cas9 protein, CRISPR-Cas9 genome directed editing vector and genome editing method | |
| CN113136374A (en) | Preparation and application of recombinant mutant Tn5 transposase | |
| CN106282173A (en) | The method of exogenous origin gene integrator site flanking sequence in cloned, transgenic biology | |
| CN113481194A (en) | DNA synthesis method | |
| CN105602972A (en) | CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors | |
| CN110747191B (en) | Polypeptide, chimeric polymerase and application thereof | |
| Sekine et al. | Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1 | |
| EP4114954A1 (en) | Method for the production of constitutive bacterial promoters conferring low to medium expression | |
| US6063595A (en) | Method of forming a macromolecular microgene polymer | |
| CN116064462A (en) | Taq DNA polymerase mutant and preparation method thereof | |
| CN110055310B (en) | Enzyme digestion based nested PCR method | |
| CN116135982A (en) | Application method and kit for gene fragment ligation | |
| Teichmann et al. | The tRNA Ser-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses | |
| Wu et al. | Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii | |
| KR100475305B1 (en) | Method for constructing chimeric dna library using exonuclease ⅶ | |
| TWI567197B (en) | Expression element, expression cassette, and vector containing the same | |
| CN108220411A (en) | A kind of PCR primer and detection method for detecting human gene CEBPA | |
| US20060199185A1 (en) | Plasmid having a function of t-vector and expression vector, and expression of the target gene using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090715 |