CN101210240B - Pointing mutation method for large primer PCR - Google Patents
Pointing mutation method for large primer PCR Download PDFInfo
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- CN101210240B CN101210240B CN2007100601554A CN200710060155A CN101210240B CN 101210240 B CN101210240 B CN 101210240B CN 2007100601554 A CN2007100601554 A CN 2007100601554A CN 200710060155 A CN200710060155 A CN 200710060155A CN 101210240 B CN101210240 B CN 101210240B
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Abstract
The invention provides a megaprimer PCR method of site-directed mutagenesis, which comprises performing a first step PCR with mutation primers and corresponding outer primers to obtain megaprimers that need not to be purified, and performing a second step PCR amplification with the megaprimers and another added outer primers to obtain target fragment. The inventive method overcomes the remarkable annealing temperature (Tm) difference of the conventional megaprimers and outer primers, improves the mutation efficiency to 100%, and is suitable for molecular biology study in laboratory.
Description
Technical field
The present invention relates to the polymerase chain reaction of rite-directed mutagenesis, particularly a kind of large primer PCR carries out the method for rite-directed mutagenesis.At first carry out the first step PCR by mutant primer and corresponding outside primer, its product is that big primer does not need purifying, takes turns pcr amplification with another outside primer initiation second of adding and goes out target fragment.The present invention has overcome conventional big primer and outside primer annealing temperature (Tm) differs significant requirement, and makes mutation efficiency reach 100%.Be fit to laboratory molecular biology method research.
Background technology
(Polymerase Chain Reaction is a kind of method of the synthetic specific DNA fragment of external enzymatic PCR), is one of the most frequently used Protocols in Molecular Biology in the polymerase chain reaction.Typical PCR is by (1) high-temperature denatured template; (2) primer and template annealing; (3) primer extends three-step reaction along template and forms a circulation, by circulating reaction repeatedly, makes target DNA be able to rapid amplification.Its key step is: template DNA to be amplified is put (be generally 93 ℃-94 ℃) under the high temperature and make its sex change separate into strand; Two Oligonucleolide primers of synthetic combine with two strands of goal gene both sides are complementary respectively under its suitable renaturation temperature, two primers on template the bonded determining positions length of amplified fragments; Heat-stable archaeal dna polymerase (Taq enzyme) begins the 3 ' end of mononucleotide from primer to mix at 72 ℃, is that template is extended from 5 ' → 3 ' direction with the goal gene, the new complementary strand of synthetic DNA.
PCR is any known goal gene of specific amplified or dna fragmentation fast, and the amplification of the goal gene in the horizontal initiate dna mixture of pik (pg) reaches the DNA fragment specific of nanogram, microgram, milligram level easily.Therefore, round pcr just is widely used for molecular biological every field rapidly once coming out.It not only can be used for separation, clone and the nucleotide sequence analysis of gene, can also be used for the structure of mutant and recombinant chou, the research of gene expression regulation, the analysis of gene pleiomorphism, the diagnosis of inherited disease and transmissible disease, the exploration of tumour mechanism, all many-sides such as legal medical expert's evaluation.Usually, PCR has following multiple use in molecular cloning and DNA analysis:
1) distinguished sequence in the generation double-stranded DNA is as probe;
2) generate the cDNA library by a small amount of mRNA;
3) some gene of clone from cDNA;
4) generate a large amount of DNA to carry out sequencing;
5) Tu Bian analysis;
6) chromosome walking;
7) dna polymorphism analyses such as RAPD, AFLP, RFLP etc.
Classical large primer PCR site-directed mutagenesis technique needs three primers, two outside forwards and reverse primer and inner mutant primer.At first carry out first round PCR by mutant primer and respective outer primer and obtain big primer, purified back is taken turns PCR with another outside primer initiation second and is obtained target fragment.In recent years, Chinese scholars improves and optimizates the large primer PCR method, makes the first step PCR product not need purifying, can directly carry out the second step PCR in a reaction tubes.But there were significant differences for the Tm of this method requirement primer, and second takes turns PCR primer Tm is higher than first round PCR primer Tm value, could get rid of the interference of low Tm value outside primer, omitted the purifying of the first step, carries out specific amplification.Yet the mutation efficiency of this method can not reach 100%.
Summary of the invention
The purpose of this invention is to provide the method that a kind of new large primer PCR carries out rite-directed mutagenesis.The significant requirement of annealing temperature gap has solved the not high shortcoming of mutation efficiency simultaneously when having broken through existing large primer PCR method to design of primers, makes it simple more, efficient, and makes mutation efficiency reach 100%, is fit to laboratory molecular biology method research.
The step that large primer PCR provided by the invention carries out the method for rite-directed mutagenesis comprises:
Forward and reverse primer of design is not subjected to the restriction of the big requirement of annealing temperature gap, and length is 24-39bp, preferred length 27-34bp, 5 ' end is introduced various restriction enzyme sites according to the original template needs, 3 ' end and template complementation, complementary base number is 9-14bp, the preferred bases radix is 9bp;
Position Design mutant primer according to the base sequence of living in that will suddenly change comprises:
The suddenly change base of rear section of original series, then mutant primer is corresponding with downstream primer, and the two causes first round PCR reaction; The suddenly change base of original series first half then should be corresponding with upstream primer, and the two causes second and takes turns the PCR reaction; The base of needs sudden changes is replaced to base after the expectation sudden change, and before and after its site, respectively extend 12bp, realize rite-directed mutagenesis;
The annealing temperature of mutant primer will be higher than forward and reverse primer, concrete step:
1) at first by the outside primer initiation first round PCR reaction of mutant primer and its correspondence, amplify the gene fragment that contains mutating alkali yl, realization is big segmental synthetic, promptly big primer;
2) product does not need purifying, directly adds another outside primer, four kinds of deoxyribonucleotides and archaeal dna polymerase in same reaction tubes, causes second and takes turns the PCR reaction, realizes the synthetic of target fragment;
Article two, the annealing temperature of outside primer and mutant primer differs 10-16 ℃.
A kind of large primer PCR carries out the method for rite-directed mutagenesis, the step that comprises:
The total length original series (IFN-α 2b) of coding human interferon-alpha-2 b be a template, 498bp, and upstream and downstream primer and 2 mutant primers of designing according to this former sequence template:
Upstream primer: 5 '-CAT AAG CTT ATG TGT GAT CTG CCT CAA-3 '
Downstream primer: 5 '-GTC GAA TTC TTC CTT ACT TCT TAA TCT-3 '
Mutant primer 1:5 '-TGT GTG ATA CAG GAG GTG GGG GTG GAG GAG ACT CCCCTG-3 '
Mutant primer 2:5 '-CTC ATG GAG GAC GCT GAT GGC CTG AGC CTT TTG GAA-3 '
1) at first causes first round PCR reaction, amplify and contain the mutator gene fragment, promptly big primer by the outside primer of mutant primer and its correspondence;
The PCR reaction system: 10 μ l Buffer damping fluids, 8 μ l dNTP, 2 μ l downstream primers, the original sequence of 2 μ l mutant primers, 1,1 μ l Interferon, rabbit is a template, 76 μ l ultrapure waters, 1 μ l pfu enzyme;
First round PCR reaction conditions: 94 ℃ of sex change 4min, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down;
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down; (24 circulations)
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 5min down;
(2) product does not need purifying, directly adds another outside primer, four kinds of thymus nucleic acids (dNTP) and archaeal dna polymerase (pfu enzyme) in same reaction tubes, causes second and takes turns the PCR reaction.
PCR reaction system: 10 μ l Buffer damping fluids, 8 μ l dNTP, 2 μ l upstream primers, 1 μ l pfu enzyme;
Second takes turns the PCR reaction conditions: 94 ℃ of sex change 40s, and 68 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (10 circulations)
94 ℃ of sex change 40s, 55 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (12 circulations)
Last 72 ℃ are extended 5min down.
Described rite-directed mutagenesis is a plurality of site mutation, and can adjacent or interval between a plurality of mutational site.
The length of described two outside primers is the 27-30 base, and melting temperature(Tm) is 55-65 ℃.
The length of described mutant primer is the 36-39 base, and melting temperature(Tm) is 70-75 ℃.
In designed mutant primer length range, catastrophe point is 1-5 a plurality of site mutations, and the suitableeest a plurality of site mutation numbers are 3-4.
Gene order after the sudden change that described method obtains.
Gene order after the sudden change of the interferon alpha 2 b that described method obtains.
Contain the plasmid pET-IFN-α 2b1 and the pET-IFN-α 2b2 of the gene order after the described IFN-α of the claim 7 2b sudden change and have Top10 (rh-IFN-α 2b1) and the transformant of Top10 (rh-IFN-α 2b2), obtain by described plasmid pET-IFN-α 2b1 and pET-IFN-α 2b2 transformed into escherichia coli competent cell respectively.
Described original template is an interferon alpha 2 b original gene sequence.
Described primer concentration is 20 μ M.
Large primer PCR novel method of the present invention has not only overcome conventional big primer and outside primer annealing temperature (Tm) differs significant requirement, has solved the not high shortcoming of mutation efficiency, makes it simple more, efficient, and makes mutation efficiency reach 100%.Be fit to laboratory molecular biology method research.
Description of drawings
Fig. 1 is the two-wheeled PCR product electrophoresis result figure of embodiment 1.
Fig. 2 is the two-wheeled PCR product electrophoresis result figure of embodiment 2.
Fig. 3 is that the enzyme of embodiment 1 is cut product electrophoresis result figure.
Fig. 4 is that the enzyme of embodiment 2 is cut product electrophoresis result figure.
Fig. 5 is that plasmid pET-IFN-α 2b makes up synoptic diagram.
Embodiment:
Embodiment 1:
Experimental procedure:
(1) the people source interferon alpha 2 b original series by National Center for Biotechnology Information (NCBI) database retrieval, utilization primer premier 5.0 software designs go out the upstream and downstream primer of this sequence, according to the base that will suddenly change, design the mutant primer 1 that contains mutating alkali yl again.
(2) outside primer (described primer concentration is 20 μ M) by mutant primer 1 and its correspondence causes first round PCR reaction, amplifies to contain the mutator gene fragment, promptly big primer.
Design upstream and downstream primer and mutant primer sequence the results are shown in Table 1.
| Mutant primer 1 | Upstream primer | Downstream primer | |
| Length (bp) | ?39 | ?27 | ?27 |
| Sequence (5 '-3 ') | ?TGT?GTG?ATA?CAG? GAG?GTG?GGG?GTG? GAG?GAG?ACT?CCC?CTG | ?CAT?AAG?GTT?ATG?TGT?GAT?CTG?CCT?CAA | ?GTC?GAA?TTC?TTG?CTT?ACT?TCT?TAA?ACT |
| Annealing temperature Tm (℃) | ?74.3℃ | ?60.3℃ | ?58.4℃ |
Underscore partly is the base after the sudden change in the table 1.CAT is the protection base in the upstream primer, and AAG CTT is a restriction enzyme site; GTC is the protection base in the downstream primer, and GAA TTC is a restriction enzyme site.
Cause first round PCR reaction by mutant primer 1 and downstream primer, reaction adds upstream primer after finishing again, and the big primer that it and last round of PCR form causes second and takes turns the PCR reaction.By annealing temperature in the table as can be known, the annealing temperature of mutant primer and upstream and downstream primer differs between 10-16 ℃, and annealing temperature differs between 0-5 ℃ between the primer of upstream and downstream.
PCR reaction system: 10 μ l Buffer damping fluid (200mM Tris-HCl, 100mM (NH
4)
2SO
4, 100mMKCl, 1%Triton X-100,20mM MgCl
2, pH 8.8), 8 μ l dNTP, 2 μ l downstream primers, 2 μ l mutant primers, 1,1 μ l interferon alpha 2 b original series is a template, 76 μ l ultrapure waters, 1 μ l pfu enzyme (Beijing ancient cooking vessel fruit Bioisystech Co., Ltd).
First round PCR reaction conditions: 94 ℃ of sex change 4min, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down;
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down; (24 circulations)
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 5min down.
Take out 5 μ l products and run EB (Ethidium bromide, ethidium bromide) electrophoresis, whether correct according to its band position and marker (DNA standard band) the big primer base number of comparatively validate synthetic.If correct, can carry out next step; Incorrect words, appropriate change reaction conditions are carried out the PCR reaction again.
(3) product does not need purifying, directly adds another outside primer, four kinds of thymus nucleic acids (dNTP) and archaeal dna polymerase (pfu enzyme) in same reaction tubes, causes second and takes turns the PCR reaction.
PCR reaction system: 6 μ l dNTP, 2 μ l upstream primers, 1 μ l pfu enzyme (the same).
Second takes turns the PCR reaction conditions: 94 ℃ of sex change 40s, and 68 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (10 circulations)
94 ℃ of sex change 40s, 55 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (12 circulations)
Last 72 ℃ are extended 5min down.
(4) after reaction finishes, take out 5 μ l products and run the EB electrophoresis, whether the base number of Interferon, rabbit sequence that contains the mutational site according to its band position and marker comparatively validate synthetic is correct.The band correct position is with PCR product purification (adopting EZ Spin Column PCR Product Purification Kit (BBI) test kit purifying).
(5) site that suddenlys change with following product test method validation.
Product test and contrast:
Interferon alpha 2 b segment and pET28C vector plasmid after the sudden change are used HindIII respectively, EcoR I double digestion, by the interferon alpha 2 b fragment after the sudden change of inserting: the above-mentioned enzyme of mixed of carrier=3: 1 is cut the product behind the purifying, with connection damping fluid (the Solution I (enzyme solution) of DNA Ligation Kit Ver 2.0 (the precious biological Dalian of TakaRa company limited) test kit) with volume, connect 1h down at 16 ℃, synthetic mutator gene fragment is inserted between the HindIII and EcoR I restriction enzyme site of plasmid pET-28C, can obtains containing the plasmid pET-IFN-α 2b1 of the interferon alpha 2 b gene order after the sudden change.To connect product is transformed in the Top10 competent escherichia coli cell under 42 ℃ of thermal shock conditions, add after 400 μ l LB liquid nutrient mediums cultivate 50min in advance, getting 200 μ l is coated on and is added with card and receives on the flat board of mycin, 37 ℃ of cultivations can obtain the transformant of called after Top10 (rh-IFN-α 2b1).Add with toothpick picking transformant after shaking table is cultivated 12h in the test tube that 5ml LB liquid nutrient medium is arranged, extract plasmid, with HindIII, EcoR I double digestion, enzyme is cut product and is verified that with 1% EB electrophoresis the result has segment at 500bp as shown in Figure 3.With the order-checking of bacterium liquid (Beijing three rich polygala root biotechnology limited liability companys), sequencing result is compared, and the mutational site is correct, accuracy rate 100%.
Embodiment 2:
Experimental procedure:
(1) with example 1, utilization primer premier 5.0 software designs go out the upstream and downstream primer of interferon alpha 2 b original series, according to the base that will suddenly change, design the mutant primer 2 that contains mutating alkali yl again.
(2) at first cause first round PCR reaction, amplify and contain the mutator gene fragment, promptly big primer by the outside primer of mutant primer and its correspondence.
Design upstream and downstream primer and mutant primer sequence the results are shown in Table 2.
Table 2
| |
Upstream primer | Downstream primer | |
| Length (bp) | 36 | ?27 | ?27 |
| Sequence (5 '-3 ') | CTC?ATG?GAG?GAC GCT?GAT? GGC? CTGAGC?CTT?TTG?GAA | ?CAT?AAG?CTT?ATG?TGT?GAT?CTG?CCT?CAA | ?GTC?GAA?TTC?TTC?CTT?ACT?TCT?TAA?ACT |
| Annealing temperature Tm (℃) | 74.6℃ | ?60.3℃ | ?58.4℃ |
Underscore partly is the base after the sudden change in the table 2.CAT is the protection base in the upstream primer, and AAG CTT is a restriction enzyme site; GTC is the protection base in the downstream primer, and GAA TTC is a restriction enzyme site.
Cause first round PCR reaction by mutant primer 2 and upstream primer, reaction adds downstream primer after finishing again, and the big primer that it and last round of PCR form causes second and takes turns the PCR reaction.By annealing temperature in the table as can be known, the annealing temperature of mutant primer and upstream and downstream primer differs between 10-16 ℃, and annealing temperature differs between 0-5 ℃ between the primer of upstream and downstream.
The PCR reaction system: 10 μ lBuffer damping fluids (the same), 8 μ l dNTP, 2 μ l upstream primers, 2 μ l mutant primers, 2,1 μ l interferon alpha 2 b original series are template, 76 μ l ultrapure waters, 1 μ l pfu enzyme (the same).
First round PCR reaction conditions: 94 ℃ of sex change 4min, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down;
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down; (24 circulations)
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 5min down.
Take out 5 μ l products and run the EB electrophoresis, whether correct according to its band position and the big primer base number of marker comparatively validate synthetic.If correct, can carry out next step; Incorrect words, appropriate change reaction conditions are carried out the PCR reaction again.
(3) product does not need purifying, directly adds another outside primer, four kinds of thymus nucleic acids (dNTP) and archaeal dna polymerase (pfu enzyme) in same reaction tubes, causes second and takes turns the PCR reaction.
PCR reaction system: 6 μ l dNTP, 2 μ l downstream primers, 1 μ l pfu enzyme (the same).
Second takes turns the PCR reaction conditions: 94 ℃ of sex change 40s, and 68 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (10 circulations)
94 ℃ of sex change 40s, 55 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; (12 circulations)
Last 72 ℃ are extended 5min down.
(4) after reaction finishes, take out 5 μ l products and run the EB electrophoresis, whether the base number of Interferon, rabbit sequence that contains the mutational site according to its band position and marker comparatively validate synthetic is correct.The band correct position with PCR product purification (the same), promptly obtains containing the interferon gene sequence in mutational site.
(5) product test and control methods are with embodiment 1.Since the mutational site difference, the interferon alpha 2 b sudden change segment difference of acquisition, and we are with the transformant called after Top10 (rh-IFN-α 2b2) that obtains in the example 2.With the order-checking of bacterium liquid (Beijing three rich polygala root biotechnology limited liability companys), sequencing result is compared, and the mutational site is correct, accuracy rate 100%.
Sequence table SEQ UENCE LISTING
＜110〉University Of Tianjin
＜120〉large primer PCR carries out the method for multidigit point rite-directed mutagenesis
<130>200707
<160>7
<170>PatentIn?version?3.4
<210>1
<211>27
<212>DNA
＜213〉Interferon, rabbit (IFN-α 2b)
<400>1
cataagctta?tgtgtgatct?gcctcaa 27
<210>2
<211>27
<212>DNA
＜213〉Interferon, rabbit (IFN-α 2b)
<400>2
gtcgaattct?tccttacttc?ttaatct 27
<210>3
<211>39
<212>DNA
＜213〉Interferon, rabbit (IFN-α 2b)
<400>3
tgtgtgatac?aggaggtggg?ggtggaggag?actcccctg 39
<210>4
<211>36
<212>DNA
＜213〉Interferon, rabbit (IFN-α 2b)
<400>4
ctcatggagg?acgctgatgg?cctgagcctt?ttggaa 36
<210>5
<211>498
<212>DNA
＜213〉Interferon, rabbit (IFN-α 2b)
<400>5
tgtgatctgc?ctcaaaccca?cagcctgggt?agcaggagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaatga 498
<210>6
<211>516
<212>DNA
＜213〉recombinant interferon (rh-IFN-α 2b)
<400>6
cataagctta?tgtgtgatct?gcctcaaacc?cacagcctgg?gtagcaggag?gaccttgatg 60
ctcctggcac?agatgaggag?aatctctctt?ttctcctgct?tgaaggacag?acatgacttt 120
ggatttcccc?aggaggagtt?tggcaaccag?ttccaaaagg?ctgaaaccat?ccctgtcctc 180
catgagatga?tccagcagat?cttcaatctc?ttcagcacaa?aggactcatc?tgctgcttgg 240
gatgagaccc?tcctagacaa?attctacact?gaactctacc?agcagctgaa?tgacctggaa 300
gcctgtgtga?tacaggaggt?gggggtggag?gagactcccc?tgatgaagga?ggactccatt 360
ctggctgtga?ggaaatactt?ccaaagaatc?actctctatc?tgaaagagaa?gaaatacagc 420
ccttgtgcct?gggaggttgt?cagagcagaa?atcatgagat?ctttttcttt?gtcaacaaac 480
ttgcaagaaa?gtttaagaag?taaggaagaa?ttcgac 516
<210>7
<211>516
<212>DNA
＜213〉recombinant interferon (rh-IFN-α 2b)
<400>7
cataagctta?tgtgtgatct?gcctcaaacc?cacagcctgg?gtagcaggag?gaccttgatg 60
ctcctggcac?agatgaggag?aatctctctt?ttctcctgct?tgaaggacag?acatgacttt 120
ggatttcccc?aggaggagtt?tggcaaccag?ttccaaaagg?ctcaggccat?cagcgtcctc 180
catgagatga?tccagcagat?cttcaatctc?ttcagcacaa?aggactcatc?tgctgcttgg 240
gatgagaccc?tcctagacaa?attctacact?gaactctacc?agcagctgaa?tgacctggaa 300
gcctgtgtga?tacagggggt?gggggtgaca?gagactcccc?tgatgaagga?ggactccatt 360
ctggctgtga?ggaaatactt?ccaaagaatc?actctctatc?tgaaagagaa?gaaatacagc 420
ccttgtgcct?gggaggttgt?cagagcagaa?atcatgagat?ctttttcttt?gtcaacaaac 480
ttgcaagaaa?gtttaagaag?taaggaagaa?ttcgac 516
Claims (1)
1. a large primer PCR carries out the method for rite-directed mutagenesis, it is characterized in that the step that comprises:
Coding human interferon-alpha-2 b amino acid whose original series IFN-α 2b is a template, 498bp, according to upstream and downstream primer and 2 mutant primers of this former sequence template design:
Upstream primer: 5 '-CAT AAG CTT ATG TGT GAT CTG CCT CAA-3 '
Downstream primer: 5 '-GTC GAA TTC TTC CTT ACT TCT TAA TCT-3 '
Mutant primer 1:5 '-TGT GTG ATA CAG GAG GTG GGG GTG GAG GAG ACT CCC CTG-3 '
Mutant primer 2:5 ' CTC ATG GAG GAC GCT GAT GGC CTG AGC CTT TTG GAA-3 '
1) at first causes first round PCR reaction, amplify and contain the mutator gene fragment, promptly big primer by the outside primer of mutant primer and its correspondence;
The PCR reaction system: 10 μ l Buffer damping fluids, 8 μ l dNTP, 2 μ l downstream primers, the original sequence of 2 μ l mutant primers, 1,1 μ l Interferon, rabbit is a template, 76 μ l ultrapure waters, 1 μ l pfu enzyme;
First round PCR reaction conditions: 94 ℃ of sex change 4min, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down;
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 30s down; 24 circulations;
94 ℃ of sex change 40s, 60 ℃ of 30s that anneal down, 72 ℃ are extended 5min down;
2) product does not need purifying, directly adds another outside primer, four kinds of thymus nucleic acid dNTP and archaeal dna polymerase pfu enzyme in same reaction tubes, causes second and takes turns the PCR reaction;
PCR reaction system: 10 μ l Buffer damping fluids, 8 μ l dNTP, 2 μ l upstream primers, 1 μ l pfu enzyme;
Second takes turns the PCR reaction conditions: 94 ℃ of sex change 40s, and 68 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; 10 circulations;
94 ℃ of sex change 40s, 55 ℃ of 25s that anneal down, 72 ℃ are extended 1min down; 12 circulations;
Last 72 ℃ are extended 5min down.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6673610B2 (en) * | 2000-08-04 | 2004-01-06 | Riken | Method for mutagenesis |
| CN101024827A (en) * | 2006-02-22 | 2007-08-29 | 中国科学院福建物质结构研究所 | Expression, purification and crystallization of urokinase catalyst structure domain mutant |
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