Method for efficiently synthesizing and fusing genes and application of method

Abstract

The invention relates to a method for efficiently synthesizing and fusing genes and application of the method. The method comprises the following steps: analyzing a to-be-synthesized and fused gene sequence, and determining a region suitable for being annealed; selecting segments with a proper length according to the region suitable for being annealed, and designing and synthesizing primers of each segment; and mixing the synthesized primers of each segment, and carrying out overlapping PCR to splice the primers of each segment, so as to obtain a spliced product, namely the synthesized and fused genes. According to the method for efficiently synthesizing and fusing the genes, the efficiency and success rate of overlapping PCR reaction can be increased, the one-time success rate can be 100% and is far higher than the one-time success rate of 70%-80% of overlapping PCR in the prior art, and tedious optimization steps of attempting PCR conditions for several times and changing the ion strength, an additive, a reaction temperature and the like in a reaction system are omitted, so that the cost is greatly lowered.

Description

technical field [0001] The invention relates to the technical field of gene synthesis, in particular to a method for efficiently synthesizing a fusion gene and its application. Background technique [0002] The current main method of gene synthesis is a two-step synthesis method. The first step is to use the PCR (polymerase chain reaction) method to splice the primers to form small fragments of 200-800bp (base pairs), which are verified by sequencing. Fragments are spliced ​​into longer desired fragments. Commonly used methods for splicing small fragments into long fragments include ligation, overlapping PCR, and in vitro recombination. The most widely used method is the overlapping PCR method. In this method, two fragments to be spliced ​​have an overlapping region, which can be used for annealing in overlapping PCR reactions. On the other two ends of the fragments, primers are designed to amplify the full length. In the PCR reaction system, one fragment can be extended ...

AI Technical Summary

Problems solved by technology

These methods do not have a clear guiding principle, and there is no clear optimization parameter to judge the direction. It is basically a trial and error process, which is time-consuming and laborious.