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Large primer PCR site-specific mutagenesis method

A technology of site-directed mutation and flanking primers, which is applied in the field of genetic engineering, can solve the problems that the mutation efficiency cannot reach 100%, and the mutation success rate is not high, and achieve the effect of shortening the annealing time and increasing the mutation rate

Pending Publication Date: 2020-04-07
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these improved methods are still affected by PCR reaction conditions, and the mutation success rate is not high after producing too long large primers, and the mutation efficiency cannot reach 100%.

Method used

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  • Large primer PCR site-specific mutagenesis method
  • Large primer PCR site-specific mutagenesis method
  • Large primer PCR site-specific mutagenesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Search and download the B1MsmE gene of Bacillus licheniformis ABC transporter through the NCBI database, and its nucleotide sequence is shown in SEQ ID NO. The lower flanking primer sequences are SEQ ID NO.2 and SEQ ID NO.3 respectively, and then it is determined that the site to be mutated is located in the first half of the target gene, and Snapgene is used to design a mutation primer 1SEQ ID NO.4 centered on the mutation site, The specific primer sequences are shown in Table 1, wherein gcagctg in the upper flanking primer sequence is a protective base, and the restriction enzyme cleavage site of the upper flanking primer is CATATG; cgac in the lower flanking primer sequence is a protective base, and the restriction enzyme cleavage site is GGATCC; the underlined portion in Table 1 represents the mutated base.

[0026] Table 1 Primer sequences

[0027]

[0028] Carry out the first round of PCR reaction according to the above mutation primers and lower flanking prim...

Embodiment 2

[0043] Search and download the B1MsmE gene of Bacillus licheniformis ABC transporter through the NCBI database, and its nucleotide sequence is shown in SEQ ID NO. The lower flanking primer sequences are SEQ ID NO.2 and SEQ ID NO.3 respectively, and then it is determined that the site to be mutated is located in the first half of the target gene, and Snapgene is used to design a mutation primer 2SEQ ID NO.7 centered on the mutation site, The specific primer sequences are shown in Table 1, wherein gcagctg in the upper flanking primer sequence is a protective base, and the restriction enzyme cleavage site of the upper flanking primer is CATATG; cgac in the lower flanking primer sequence is a protective base, and the restriction enzyme cleavage site is GGATCC; the underlined portion in Table 1 represents the mutated base.

[0044] Table 2 Primer sequences

[0045]

[0046] Carry out the first round of PCR reaction according to the above mutation primers and lower flanking prim...

Embodiment 3

[0061] Search and download the B1MsmE gene of Bacillus licheniformis ABC transporter through the NCBI database, and its nucleotide sequence is shown in SEQ ID NO. The lower flanking primer sequences are SEQ ID NO.2 and SEQ ID NO.3 respectively, then determine that the site to be mutated is located in the first half of the target gene, and use Snapgene to design a mutation primer 3SEQ ID NO.10 centered on the mutation site, The specific primer sequences are shown in Table 1, wherein gcagctg in the upper flanking primer sequence is a protective base, and the restriction enzyme cleavage site of the upper flanking primer is CATATG; cgac in the lower flanking primer sequence is a protective base, and the restriction enzyme cleavage site is GGATCC; the underlined portion in Table 1 represents the mutated base.

[0062] Table 3 Primer sequences

[0063]

[0064] Carry out the first round of PCR reaction according to the above mutation primers and lower flanking primers, wherein t...

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Abstract

The invention provides a large primer PCR site-specific mutagenesis method, which comprises the following operation steps: (1) designing primers according to a target gene sequence to obtain two flanking primers which are an upper flanking primer and a lower flanking primer; (2) designing a mutation primer according to the sequence position of the mutation base, wherein the mutation site is positioned in the middle of the mutation primer sequence; (3) carrying out a first-step PCR reaction on the mutation primer and the flanking primer far away from the mutation site to obtain a large primer;and (4) adding two flanking primers into the obtained large primer to carry out second-step PCR amplification so as to obtain a mutant target gene sequence. According to the method, when the mutationprimer is designed, the mutation site is designed in the middle of a mutation primer sequence, so that the mutation primer is favorably combined with a template in the subsequent PCR reaction, and themutation rate is improved.

Description

technical field [0001] The present disclosure relates to the technical field of genetic engineering, in particular to a method for site-directed mutagenesis by mega-primer PCR. Background technique [0002] Site-directed mutagenesis (SDM) is an indispensable research method to elucidate the relationship between gene expression and protein structure and function. Polymerase chain reaction (Polymerase Chain Reaction, PCR) is a molecular biology technique used to amplify specific DNA fragments in vitro, which can greatly increase the trace amount of DNA. The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. The PCR reaction consists of three basic reaction steps: denaturation-annealing-extension: after the template DNA is heated at high temperature (95℃~98℃) for a certain period of time, the double-stranded DNA or the double-stranded D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/102
Inventor 钟先锋杨思婷苏思韵张宇博黄珍金吕蕾
Owner FOSHAN UNIVERSITY
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