A gene mutation detection method and reagent for reducing amplification bias

Abstract

The invention discloses a gene mutation detection method for reducing amplification bias and a reagent. The method comprises the following steps: (a) performing first-step one-way guide extension of a joint connection product containing the target area DNA by use of a first-step one-way guide extension primer, and performing PCR amplification; (b) performing second-step one-way guide extension of the product of the step (a) by use of a second-step one-way guide extension primer, and performing PCR amplification; (c) modifying the product of the step (b), and treating the product of the step (b) with duplex-specific nuclease at a temperature suitable for annealing the modified product of the step (b); and (d) performing PCR amplification of the product of (c) by use of a second universal primer and a third universal primer and sequencing. Through the method, the content of high-abundance DNA is close to that of low-abundance DNA, an aim of reducing amplification bias is achieved, and the low-abundance gene mutation is accurately detected at low cost through a high-throughput sequencing technology.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a gene mutation detection method and a reagent for reducing amplification bias. Background technique [0002] Genetic testing is a technology that detects a subject's DNA through blood, other bodily fluids or cells. Genetic testing can diagnose diseases and can also be used to predict the risk of diseases. Early disease screening, disease diagnosis, and concomitant treatment through genetic testing often require accurate and sensitive detection of multiple low-abundance gene mutation sites. For example, tumors are highly heterogeneous in which disease-causing mutations may be present in very low proportions. To achieve precise gene detection of tumors, it is necessary to find relevant low-abundance gene mutations from a large amount of non-tumor DNA and tumor DNA with non-pathogenic mutations. [0003] The commonly used gene detection methods are all based on PCR (polym...

AI Technical Summary

Problems solved by technology

However, reducing the number of PCR cycles will directly lead to a low yield of amplification products, especially when the amount of DNA material to be detected is very small, it cannot produce enough PCR products for detection, and it is difficult for low-abundance DNA mutations to achieve sufficient copy numbers for use. for detection

Another solution is to use a DNA polymerase with superior amplification uniformity. This solution can only reduce the amplification bias to a small extent, and often cannot solve practical problems.

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