Rapid bidirectional multilocus gene mutation method

A multi-site mutation and multi-site technology, applied in the field of bioengineering, can solve the problems of long time and limited mutation sites.

Inactive Publication Date: 2012-09-26
王书崎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR-induced site-directed mutagenesis includes overlap extension mutation and one-step rapid mutation, etc. It is accurate and efficient. Among them, overlap extension mutation PCR can achieve site-directed mutation at any position in the DNA segment, but it takes a long time and the mutation sites are limited.

Method used

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  • Rapid bidirectional multilocus gene mutation method
  • Rapid bidirectional multilocus gene mutation method
  • Rapid bidirectional multilocus gene mutation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Obtaining a 324 bp DNA fragment containing two mutations

[0013] 1. Design of primers

[0014] According to the quasi-mutant site and its sequence characteristics of the selected DNA fragment (see sequence table SEQ ID No. 1), three pairs of specific primers were designed to amplify the selected DNA fragment and its adjacent upstream and downstream fragments ( F1: upstream fragment, F2: target sequence, F3: downstream fragment). The designed primer sequences are shown in Table 1, where the underlined part is the extended sequence, that is, the overlapping part.

[0015] Table 1 Primer sequences

[0016] name Primer sequence (5'-3') Length (nt) F1F AGATCTGAGCCTGGGAG 17 F1RT GGTTCCGTTATTTCCCAAGTT TTAAGCAGTGGGTTCCCTAG 40 F2FT AACTTGGGAATAACGGAACC GAGTGCTTCACTTGATACAG 40 F2RT GCCAATCTCTCAATCGCGGC ATTTTCCACACTGACTAAAAG 41 F3FT GCCGCGATTGAGAGATTGGC CAGGGACCTGAAAGCGAAAG 40 F3R CACCCATCTC...

Embodiment 2

[0023] Embodiment 2: the length is 324 bp, contains the acquisition of two mutant DNA fragments

[0024] In this example, except for the template and primers, the two rounds of PCR amplification conditions are the same as in Example 1. The final product is also 324bp in length and contains a point mutation sequence at both ends. The primers are shown in Table 2, and the underlined part indicates the overlapping part. For the mutation product, see SEQ. ID. No.13 in the sequence listing.

[0025] Table 2 Primer sequences

[0026] name Primer sequence (5'-3') Length (nt) F1F AGATCTGAGCCTGGGAG 17 F1RT 4m GGTACCGTTATTTCCCAAGTT TTAAGCAGTGGGTTCCCTAG 40 F2FT 4m AACTTGGGAATAACGGTACC GAGTGCTTCACTTGATACAG 40 F2RT 4m GCCAATCTCTCCAATCCCGGC ATTTTCCACACTGACTAAAAGG 42 F3FT 4m GCCGGGATTGAGAGATTGGC CAGGGACCTGAAAGCGAAAG 40 F3R CACCCATCTCTCTCCTTCTA 20

[0027] Wang Shuqi

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Abstract

The invention relates to a rapid bidirectional multilocus gene mutation method. According to the method, by taking an improved overlap extension PCR method as a basis and adopting sequence specific primers with mutation sites, DNA bidirectional multilocus gene mutation is realized by carrying out two PCR amplifications and joining DNA segments. The method can be widely used in target gene site-directed mutagenesis, gene synthesis, protein structure-function research, protein expression optimization and target gene packaging. According to the method, three DNA segments and three corresponding pairs of specific amplification primers are designed based on sequence characteristics of DNA segments needing mutation. Target gene segments for introducing needed mutation sites are formed by carrying out two PCR amplifications and setting specific amplification programs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a fast and efficient gene mutation method. The method is based on an improved overlap extension PCR method, utilizes specific primers with mutation sites, and realizes DNA bidirectional multi-site gene mutation through two rounds of PCR amplification and DNA fragment connection. Background technique [0002] With the continuous development of molecular biology research, gene site-directed mutagenesis technology has become a commonly used method for modifying and optimizing genes, and has been widely used in the research of gene synthesis, gene expression and the relationship between protein structure and function. . PCR-mediated point mutation is the most commonly used gene site-directed mutagenesis technique. PCR-induced site-directed mutagenesis includes overlap extension mutation and one-step rapid mutation, which are accurate and efficient. Among them, ov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王书崎隗慧林徐峰
Owner 王书崎
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