Construction method of amplicon library for detecting low-frequency mutation of target gene

Abstract

The invention discloses a construction method of an amplicon library for detecting low-frequency mutation of a target gene. The construction method of the amplicon library for detecting the low-frequency mutation of the target gene, provided by the invention, comprises the following steps: 1) designing and synthesizing a Barcode primer F1, an upstream primer F2, a downstream outer primer R1 and adownstream inner primer R2; 2) carrying out further PCR (Polymerase Chain Reaction) amplification on a cfDNA (cell-free Deoxyribonucleic Acid) of a sample to be detected by utilizing the Barcode primer F1, the upstream primer F2, the downstream outer primer R1 and the downstream inner primer R2, so as to obtain an amplified product, namely a DNA library for amplicon sequencing. By adopting the method disclosed by the invention, a tissue sample can be detected and different regions of free DNAs in samples including blood, urine, cerebrospinal fluid and the like can be rapidly, conveniently, sensitively and specifically subjected to target amplification and mutation which is as low as a 0.1 percent level is efficiently detected; the experiment operation is greatly simplified, the library loss and pollution are effectively avoided, the cost is remarkably reduced and the efficiency is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing an amplicon library for detecting low-frequency mutations of target genes. Background technique [0002] Tumors are highly heterogeneous, and the pathogenic mutations in them may exist in a very low proportion. The mutation frequency of the mutation site or mutation region of the target gene in the cfDNA in the blood, urine and cerebrospinal fluid of tumor patients will affect the future of tumors. Judgment on drug use or tumor development direction. Therefore, detecting the mutation frequency of the mutation site or mutation region of the target gene in the cfDNA in the blood, urine and cerebrospinal fluid of tumor patients has become a research focus, which requires sequencing of the mutation site or mutation region to detect the mutation frequency. [0003] At present, the error rate of Hiseq sequencing, the most accurate next-generation sequ...

AI Technical Summary

Problems solved by technology

However, the conventional library construction methods on the market have defects such as high cost, long detection cycle, complicated process, easy contamination of the library, and high requirements for testing personnel, and are not suitable for sequencing library construction of a large number of samples

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