Gene mutation, deletion, insertion and recombinant fragment obtaining method based on overlapping extension PCR process

Abstract

The invention relates to a gene mutation, deletion, insertion and recombinant fragment obtaining method based on an overlapping extension PCR process. The method comprises the following steps: designing two or three pairs of primers, wherein adjacent fragment primers contain a 1bbp overlapping sequence at the tail, and an outer primer contains an enzyme digestion site at the tail; carrying out parallel complete PCR reaction by utilizing the designed primers and adding the to-be-fused gene fragment or plasmid DNA as a template, thereby obtaining two or three groups of PCR products containing 15 overlapping basic groups at a fusion site; taking a PCR product containing a target fragment as a template, adding one pair of outside primers of the to-be-fused full-length gene, and carrying out secondary PCR reaction, so that the to-be-fused target gene is obtained. The gene mutation, deletion, insertion and recombinant fragment obtaining method provided by the invention has the characteristics of simple operation, rapidness and high efficiency and can be used for solving the problems that the conventional SOE-PCR overlapping primer is relatively long, too high annealing temperature and unnecessary waste during primer synthesis can not be caused, no stripe is produced during electrophoresis of the PCR product sometime and a dispersion zone and a non-specific stripe can be easily produced.

Description

technical field [0001] The invention relates to a method for obtaining gene mutation, deletion, insertion and recombination fragments, in particular to obtaining gene mutation, deletion, insertion and recombination fragments by using the overlapping extension PCR method. Background technique [0002] Mutation, deletion, insertion, and recombination of genes are often required in the study of gene function. Although there are many available kits for this purpose, most of them are expensive and require high operating techniques. Since the overlapping extension PCR technique (splicing by overlap extension PCR, SOE-PCR) was reported by Horton et al. in 1989, this technique has been perfected and developed and widely used. Overlap extension PCR uses primers that are complementary to the 5' terminal base at the fusion site to denature and anneal the PCR product to form a complementary chain of the overlapping part. The augmented fragments are spliced ​​accurately and completely, ...

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Problems solved by technology

[0003] However, when we constructed the fusion gene by overlap extension PCR, we found that if the overlap primers are too long, the annealing temperature will be too high and unnecessary synthesis will be wasted. Sometimes there will be no bands in PCR, and it is difficult to obtain the expected results.

Although Meng Xiangping invented the overlap extension PCR method (CN 103820430A) of DNA fragments with high GC content or complex structure DNA as a template, it has improved the long time-consuming, high number of cycles and complicated process of conventional overlap extension PCR. However, the process products in the middle of this method cannot be detected, and the primers, annealing temperature and PCR conditions cannot be determined whether they are suitable. Sometimes there is no band in SOE-PCR, and the reason is difficult to judge

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