Fast high-flux gene site-directed mutagenesis method

Abstract

The invention provides a gene fixed-point induction method of high speed and high throughput, which comprises the following procedures: 1. constructing a bacterial strain expressing DNA adenine methylate enzyme shortcoming of Dpnl limiting incision enzyme; 2. analyzing the sequence of the position to be varied in the gene as necessary, designing partially overlapped (10-16 nt) PCR inducing primer; 3. expanding reversely PCR; 4. transforming directly in a chemical way by the expanded PCR product the constructed bacterial strain for expressing DNA adenine methylate enzyme shortcoming of Dpnl limiting incision enzyme. In this way, plasmid with preset variant is got by Dpnl in-body sieving. The invention can truly carry out fixed-point induction in a simple and rapid way without ordering special decorative primer, and can rapidly and easily construct varied plasmid without any treatment over PCR output (enzyme connection, out-body hybridizing, rubber recovering and out-body Dpnl digesting, etc.).

Description

technical field [0001] The invention relates to gene manipulation technology, in particular to a rapid and high-throughput PCR site-directed mutagenesis method for gene modification. It is especially suitable for constructing mutants such as gene deletion, insertion and point mutation to study the function of genes. Background technique [0002] With the completion of the genome projects of various species including the human genome project, the development of a simple and rapid site-directed mutagenesis technique to study the functions of genes and proteins has received urgent attention. At present, some companies and research institutes have developed gene site-directed mutagenesis methods. Due to the simplicity and ease of operation of PCR, the method of site-directed mutagenesis based on PCR has been favored by many researchers. Site-directed mutagenesis based on PCR mainly includes overlap extension PCR method, large primer method and inverse PCR method, etc. The fir...

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Problems solved by technology

However, this method uses two perfectly matched primers, which have the following disadvantages: (1) the PCR amplification efficiency is low; (2) the PCR reaction has a large dependence on the quality and quantity of the template plasmid, which is shown in the need to prepare A relatively pure plasmid and a relatively large number of templates are added in the PCR reaction; (3) PCR amplification efficiency and mutagenesis efficiency are easily affected by the size of the plasmid vector, which is not easy to amplify and compare the mutagenesis efficiency of larger plasmid vectors. (4) In addition, this method is more effective for point mutations, but it is difficult to introduce large gene insertion and deletion mutations, which limits the wide application of this method

Although the method of circularizing PCR products based on homologous recombination in vivo in Escherichia coli has simple steps, this method uses a relatively long (25nt) primer overlapping region when designing mutagenic primers, and needs to introduce PCR products into Escherichia coli by electroporation In addition, it needs to optimize the reaction conditions of PCR to obtain relatively high mutagenesis efficiency

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