CN107974450A - A kind of PCR Efficient amplification methods of the long sequence gene of no base mutation - Google Patents
A kind of PCR Efficient amplification methods of the long sequence gene of no base mutation Download PDFInfo
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- CN107974450A CN107974450A CN201711499310.2A CN201711499310A CN107974450A CN 107974450 A CN107974450 A CN 107974450A CN 201711499310 A CN201711499310 A CN 201711499310A CN 107974450 A CN107974450 A CN 107974450A
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
This application discloses a kind of PCR Efficient amplification methods of the no long sequence gene of base mutation, it is characterised in that amplification system includes：10×Buffer for KOD‑Plus‑Ver.2：5,2mM dNTPs：5,25mM MgSO4：3, Primer F (10 μM)：1.5, Primer R (10 μM) 1.5, Template DNA：1, KOD Plus Neo：1, ddH2O complements to 50.PCR response procedures：94 DEG C of pre-degenerations, 2min；98 DEG C of denaturation, 10sec；60 DEG C of annealing, 30sec；68 DEG C of extension, 1min；4 circulations；Extend 68 DEG C afterwards, 10min；4 DEG C of insulations.The method of the present invention, which can improve, obtains accurate genetic fragment, provides safeguard for follow-up gene function.
Description
Technical field
It is more particularly to a kind of efficiently to obtain the length without base mutation the present invention relates to genetic engineering molecule clone technology field
The method of PCR amplification and the TA clone of sequence gene.
Background technology
Base species and to put in order be the supporting body of biological heredity information.In vivo, proofreaded during DNA replication dna
(proofreading) process is very stringent, avoids mispairing there are various mechanism in vivo and repairs mispairing.
With the development of biotechnology, outer-gene amplification technique is gradually improved, and outer-gene amplification is based on archaeal dna polymerase
Chain type chain reaction.But often due to primer concentration, annealing temperature, template purity, circulation time during expanding in vitro
Number and archaeal dna polymerase, which lack 3 ' -5 ' 5 prime excision enzyme activities, causes base mispairing.
Longer with the purpose fragment of amplification, DNA polymerase activity fails more serious, the possibility of base mispairing occurs
It is bigger.It is often missense mutation after the base mutation occurred in amplification in vitro, so as to draw although codon has degeneracy
The change of amino acid is played, and then influences protein function, therefore in the research of gene cloning and gene function analysis, avoids alkali
Base mutation is the technical problem of overriding concern.
But external PCR but lacks these processes, thus in organism DNA replication dna mismatch rate it is more much lower than PCR,
Being carried out in vitro using round pcr in amplification procedure, accurate match of base depends on used archaeal dna polymerase, and one
As archaeal dna polymerase mismatch rate about 1 × 10-5/bp.And the archaeal dna polymerase of pfu classes carries the enzyme of 5 prime excision enzyme activity, mismatch rate
It is more much lower than Taq enzyme, therefore select that mispairing occur also related with period in the present invention.
The content of the invention
The technical problems to be solved by the invention are, there is provided a kind of PCR of the no long sequence gene of base mutation is efficient
Amplification method.The method of the present invention carries out the amplification of EAT1 genes using hi-fi, have selected 10 repetitions.In the process of amplification
In we used independent development a kind of PCR patterns, while difference has been carried out to result as compareing using conventional PCR method
Specific analysis.
In order to solve the above technical problems, the present invention provides a kind of PCR efficient amplifications of the no long sequence gene of base mutation
Method, amplification system include：
Amplification program includes：PCR response procedures：94 DEG C of pre-degenerations, 2min；98 DEG C of denaturation, 10sec；60 DEG C of annealing,
30sec；68 DEG C of extension, 1min；4 circulations；Extend 68 DEG C afterwards, 10min；4 DEG C of insulations.
It may further include：Previous PCR product is diluted 10 times, takes PCR solution that 1 μ l have diluted and 3ul
Template DNA solution, takes templates of the 1 μ l as PCR amplification next time, amplification system and amplification program are respectively as weighed after mixing
Profit is required described in 1 and claim 2.
It may further include：Repeat to operate 3 times according to claim 3 the method, the PCR that 3 operations obtain is produced
Thing mixes, agarose electrophoresis PCR product, and gel extraction is carried out to purpose fragment.
It may further include：PCR recovery products are added into A tails.
It is described to add the reaction system of A tails to include PCR recovery products：
System will be prepared and be placed in 72 DEG C of metal bath 30min.
It may further include：The TA clones of PCR product：Purpose fragment after tailing is connected to Takara companies
On PMD-18T, 16 DEG C, connection overnight.
TA clone's systems of the PCR product include：
It may further include：The conversion of bacillus coli DH 5 alpha competent cell.
Further comprise：Picking single bacterium colony carries out bacterium colony PCR detections, purpose fragment size and expected clip size coincide into
The homologous result for comparing analysis, choosing homology 100% of row.
Beneficial effect of the present invention includes：The present invention is one based on the accuracy exploitation for improving gene long segment PCR amplification
Kind PCR patterns, can improve and obtain accurate genetic fragment, provide safeguard for follow-up gene function.
Brief description of the drawings
Fig. 1 is the normal PCR amplification electrophoretogram of EAT1 expression casettes described in the embodiment of the present invention；
Fig. 2 is that TA cloning vectors described in the embodiment of the present invention convert E. coli competent photo.
Embodiment
The present invention is described in detail with reference to embodiment.To make the objects, technical solutions and advantages of the present invention clearer, bright
Really, developing simultaneously referring to the drawings, the present invention is described in more detail for embodiment, but the invention is not limited in these embodiments.
Unless otherwise instructed, the raw material in the embodiment of the present invention and catalyst are bought by commercial sources.
In the present invention, we expand the complete gene orders of rice EAT1, including the promoter of EAT1 genes and
Terminator, EAT1 genes all extron and introne, complete EAT1 expression casettes length are 5225bp.Pass through EAT1
The base structure of gene is analyzed, and the GC% about 40% of the expression cassette of the gene, there is no the problem of high GC.The present invention is implemented
We have selected Japan spinning KOD-Plus-Neo to example, and the amplification of EAT1 genes is carried out using its hi-fi, have selected 10 weights
It is multiple.We used a kind of PCR patterns of independent development during amplification, while using conventional PCR method as control,
Difference analysis has been carried out to result.
In order to solve the above technical problems, the present invention provides a kind of PCR efficient amplifications of the no long sequence gene of base mutation
Method, amplification system include：
Amplification program includes：PCR response procedures：94 DEG C of pre-degenerations, 2min；98 DEG C of denaturation, 10sec；60 DEG C of annealing,
30sec；68 DEG C of extension, 1min；4 circulations；Extend 68 DEG C afterwards, 10min；4 DEG C of insulations.
It may further include：Previous PCR product is diluted 10 times, takes PCR solution that 1 μ l have diluted and 3ul
Template DNA solution, takes templates of the 1 μ l as PCR amplification next time, amplification system and amplification program are respectively as weighed after mixing
Profit is required described in 1 and claim 2.
It may further include：Repeat to operate 3 times according to claim 3 the method, the PCR that 3 operations obtain is produced
Thing mixes, agarose electrophoresis PCR product, and gel extraction is carried out to purpose fragment.
It may further include：PCR recovery products are added into A tails.
It is described to add the reaction system of A tails to include PCR recovery products：
System will be prepared and be placed in 72 DEG C of metal bath 30min.
It may further include：The TA clones of PCR product：Purpose fragment after tailing is connected to Takara companies
On PMD-18T, 16 DEG C, connection overnight.
TA clone's systems of the PCR product include：
It may further include：The conversion of bacillus coli DH 5 alpha competent cell.
Further comprise：Picking single bacterium colony carries out bacterium colony PCR detections, purpose fragment size and expected clip size coincide into
The homologous result for comparing analysis, choosing homology 100% of row.
1st, according to rice full-length genome data, the complete sequence of EAT1 expression casettes is obtained, utilizes Primer5.0 softwares
Primer is designed, primer sequence is (EAT1-F/EAT1-R)
2nd, rice leaf DNA is extracted using CTAB methods, extraction step is as follows：
3rd, using rice leaf genomic DNA as template, EAT1 expression casettes are carried out by EAT1-F/EAT1-R primers
Amplification, amplification system and amplification program are as follows：
Amplification system
1 KOD-Plus-Neo amplification systems of table
Amplification program：PCR response procedures are：94 DEG C of pre-degenerations, 2min；98 DEG C of denaturation, 10sec；60 DEG C of annealing, 30sec；
68 DEG C of extension, 1min；4 circulations；Extend 68 DEG C afterwards, 10min；4 DEG C of insulations.
4th, above-mentioned PCR product is diluted 10 times, takes PCR solution that 1 μ l have diluted and the template DNA solution of 3ul, mixed
Templates of the 1 μ l as PCR amplification next time is taken after closing uniformly, amplification system and amplification program are as described in 3.
5th, the PCR product in 4 is diluted 10 times, takes PCR solution that 1 μ l have diluted and the template DNA solution of 3ul,
Take templates of the 1 μ l as PCR amplification next time after mixing, amplification system and amplification program are still as described in 3.
6th, the PCR product obtained in 3,4,5 is mixed, agarose electrophoresis PCR product, purpose fragment is carried out to cut glue
Recycling, specific steps refer to the operation instructions in OMEGA plastic recovery kits.
7th, PCR recovery products are added into A tails, reaction system is shown in Table 2-2.
2 PCR product of table adds A end reaction systems
System will be prepared and be placed in 72 DEG C of metal bath 30min.
8th, carrier T is connected.Purpose fragment after tailing is connected on the PMD-18T of Takara companies, 16 DEG C, connected overnight
Connect.TA clone's systems of PCR product are shown in Table 2-3,
The TA clones of 3 PCR product of table
9th, the conversion of bacillus coli DH 5 alpha competent cell
10th, picking single bacterium colony carries out bacterium colony PCR detections, and purpose fragment size and the carry out that expected clip size is coincide are follow-up
Sequencing analysis
11st, sequencing result and known EAT1 sequences are carried out it is homologous compare analysis, the result of selection homology 100% into
The experiment of row next step.
As a result：Using KOD-Plus-Neo carry out Standard PCR, in cyclic process due to repeatedly experience denaturation, annealing,
Extension and renaturation, the decline of archaeal dna polymerase enzymatic activity, amplified fragments are longer, and the extension of time of archaeal dna polymerase experience is longer, with
The increase of cycle-index, base pairing accuracy are reduced and become apparent from, the probability increase that mispairing occurs in base or base is lost.Therefore
Conventional method PCR amplification, 10 repetition experiments have the mispairing of base or the Loss of base.And under new PCR patterns, pass through
PCR cycle number is reduced, and by updating archaeal dna polymerase and supplementing the original DNA profiling in part, is cloned subsequently through TA, greatly
The conversion of enterobacteria competence, single bacterium colony PCR detections and follow-up sequencing analysis.Occurring two amplified fragments in 10 single bacterium colonies is
The right-on single bacterium colony of sequence of 5225bp, reaches follow-up requirement of experiment.
4 Standard PCR pattern of table is counted with base mutation after EAT1 expression casettes amplification under new PCR patterns and deletion segment
The above, is only several embodiments of the present invention, any type of limitation is not done to the present invention, although this hair
It is bright with preferred embodiment disclose as above, but and be not used to limitation the present invention, any person skilled in the art, is not taking off
In the range of technical solution of the present invention, make a little variation using the technology contents of the disclosure above or modification is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection domain.
Claims (10)
1. a kind of PCR Efficient amplification methods of the long sequence gene of no base mutation, it is characterised in that amplification system includes：
2. the PCR Efficient amplification methods according to claim 1 without the long sequence gene of base mutation, it is characterised in that amplification
Program includes：PCR response procedures：94 DEG C of pre-degenerations, 2min；98 DEG C of denaturation, 10sec；60 DEG C of annealing, 30sec；68 DEG C of extension,
1min；4 circulations；Extend 68 DEG C afterwards, 10min；4 DEG C of insulations.
3. the PCR Efficient amplification methods according to claim 2 without the long sequence gene of base mutation, it is characterised in that into one
Step includes：Previous PCR product is diluted 10 times, takes PCR solution that 1 μ l have diluted and the template DNA solution of 3ul, mixing
Templates of the 1 μ l as PCR amplification next time is taken after uniformly, amplification system and amplification program are respectively as claim 1 and right will
Ask described in 2.
4. the PCR Efficient amplification methods according to claim 3 without the long sequence gene of base mutation, it is characterised in that into one
Step includes：Repeat to operate 3 times according to claim 3 the method, the PCR product that 3 operations obtain is mixed, agarose electrophoresis
PCR product, gel extraction is carried out to purpose fragment.
5. the PCR Efficient amplification methods without the long sequence gene of base mutation according to any one of Claims 1 to 4, it is special
Sign is, further comprises：PCR recovery products are added into A tails.
6. the PCR Efficient amplification methods according to claim 5 without the long sequence gene of base mutation, it is characterised in that described
The reaction system that PCR recovery products add A tails is included：
System will be prepared and be placed in 72 DEG C of metal bath 30min.
7. the PCR Efficient amplification methods without the long sequence gene of base mutation according to any one of claim 1~6, it is special
Sign is, further comprises：The TA clones of PCR product：Purpose fragment after tailing is connected to the PMD-18T of Takara companies
On, 16 DEG C, connection overnight.
8. the PCR Efficient amplification methods according to claim 7 without the long sequence gene of base mutation, it is characterised in that described
TA clone's systems of PCR product include：
9. the PCR Efficient amplification methods without the long sequence gene of base mutation according to any one of claim 1~8, it is special
Sign is, further comprises：The conversion of bacillus coli DH 5 alpha competent cell.
10. the PCR Efficient amplification methods according to claim 9 without the long sequence gene of base mutation, it is characterised in that into one
Step includes：Picking single bacterium colony carries out bacterium colony PCR detections, and purpose fragment size is coincide with expected clip size carries out homologous compare point
Analysis, chooses the result of homology 100%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632130A (en) * | 2003-12-22 | 2005-06-29 | 中国科学院成都生物研究所 | Long distance PCR amplification method |
| CN104911194A (en) * | 2015-06-04 | 2015-09-16 | 山东农业大学 | Wheat male sterility genes WMS and application of anther specific promoter thereof |
| CN105754992A (en) * | 2016-04-05 | 2016-07-13 | 南昌大学 | Method for conducting efficient PCR amplification on long-fragment target sequence |
-
2017
- 2017-12-31 CN CN201711499310.2A patent/CN107974450A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632130A (en) * | 2003-12-22 | 2005-06-29 | 中国科学院成都生物研究所 | Long distance PCR amplification method |
| CN104911194A (en) * | 2015-06-04 | 2015-09-16 | 山东农业大学 | Wheat male sterility genes WMS and application of anther specific promoter thereof |
| CN105754992A (en) * | 2016-04-05 | 2016-07-13 | 南昌大学 | Method for conducting efficient PCR amplification on long-fragment target sequence |
Non-Patent Citations (3)
| Title |
|---|
| TOYOBO公司: "《KOD-Plus-》", 《百度文库》 * |
| TOYOBO公司: "《KOD-Plus-Neo》", 《百度文库》 * |
| 唐炳华: "《聚合酶链反应》", 《分子生物学》 * |
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Application publication date: 20180501 |