High-efficiency and fidelity PCR method for fixed-point introduction of unpaired regions

Abstract

The invention provides a high fidelity polymerase chain reaction (PCR) method for a fixed point introduction mismatching area. According to the high fidelity two-step PCR method for the fixed point introduction mismatching area, polymerases with different properties are respectively used, and the PCR is carried out in a respective optimized buffer system. The method comprises the following steps: carrying out the PCR by using a tag enzyme with low fidelity and high amplification capacity, so that the problem that a primer is not matched with a template is solved, thereby obtaining a product with an area which is not matched with the original template (enzyme cutting site-containing primer area or point mutation area); and completely matching a special high-fidelity Pfu enzyme for performing amplification by taking the product in the previous step as a template. Therefore, the problems of amplification efficiency and high fidelity in the PCR reaction with the mismatching area are well solved.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a PCR amplification method, in particular to a PCR method for introducing an unpaired region with a template at a designated site. Background technique [0002] In the PCR process of site-specific introduction of unpaired regions, such as PCR and site-directed mutagenesis, which add restriction sites at the 5' end of primers, are commonly used techniques in molecular biology, and have extremely high requirements for the PCR process: the original paired regions require high fidelity , while the site-specific unpaired region requires the ability to achieve directional mutation and the product requires high-efficiency amplification. However, it is difficult for experimental operators to balance fidelity, specific mutation and efficiency in the actual operation process; in addition to factors such as primer design and annealing temperature, the concentration of polymerase and Mg2+ used ...

AI Technical Summary

Problems solved by technology

However, the proportion of Pfu in the mixed enzyme that emphasizes fidelity is high, and the existence of the primer mismatch region makes it always play a 3’ → 5’ exonuclease activity, and the low concentration of Mg 2+ It greatly affects the amplification efficiency of tags, and the amplification efficiency of unpaired products is far lower than that of tag enzymes; the proportion of Tag in mixed enzymes that emphasize amplification efficiency increases, and the error rate is high, so the high concentration of Mg used 2+ Also reduces the specificity of Pfu

The success rate of this mixed enzyme to obtain the target gene sequence in the PCR experiment of site-specific introduction of unpaired regions is actually not high

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