Method for carrying out blocking, substitution, amplification, enrichment and detection on target mutation based on blocker introducing extra base mismatch

Abstract

The invention relates to a method for enriching and detecting gene mutation of a target area. The method for enriching and detecting the gene mutation of the target area comprises the following steps:mixing a nucleic acid sample containing a to-be-detected target area with a first primer, a second primer and a blocking and amplification sequence, and carrying out first PCR amplification to obtaina first amplification product; and carrying out second PCR amplification by utilizing the first primer and the second primer based on the first amplification product to obtain a second amplificationproduct, wherein the 3' tail end of the blocking and amplification sequence contains a blocking group, the blocking and amplification sequence and the first primer contain a partial overlapping sequence, the to-be-detected target area is located in a matching area of the blocking and amplification sequence and the nucleic acid sample, the 3' tail end of the first primer is located at the upstreamof the to-be-detected target area, at least one base mismatching site exists between the blocking and amplification sequence and the nucleic acid sample, and the at least one base mismatching site isdifferent from a site of the to-be-detected target area. Therefore, high-precision detection for low-frequency gene mutation can be realized.

Description

technical field [0001] The present invention relates to the field of gene sequencing, in particular to a method for enriching and detecting gene mutations in a target region, and in particular to a method for detecting target mutations by blocking, replacing, amplifying, and enriching based on the introduction of additional base mismatch blockers Methods. Background technique [0002] With the rapid development of the times and technology, the concept of cancer treatment is undergoing fundamental changes. The International Union Against Cancer believes that 1 / 3 of cancers can be prevented, 1 / 3 of cancers can be cured if diagnosed early, and 1 / 3 of cancers can be treated correctly to relieve pain and prolong life. The treatment of cancer has also changed from empirical science to evidence-based medicine, from cell attack mode to targeted treatment mode. Early traditional treatment methods were surgical resection, chemotherapy and radiotherapy, among which chemotherapy and r...

AI Technical Summary

Problems solved by technology

Therefore, there is an urgent need for a ctDNA detection technology that can not only detect low-frequency mutation sites but also achieve a certain throughput. None of the main ctDNA detection technologies on the market can meet the requirements.

This kind of low-frequency mutation site detection with a certain throughput is a bottleneck encountered by current ctDNA detection technology

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