Ultra-low frequency mutant nucleic acid fragment detection method, library construction method, primer design method and reagent

Abstract

The invention discloses an ultra-low frequency mutant nucleic acid fragment detection method, a mutant nucleic acid fragment library construction method, a primer design method and a reagent. Firstly,the fragmented nucleic acid of the sample to be tested is subjected to terminal repair, end-filling and adding A; connecting molecular tag joints; using the combination of molecular tag primers and single molecule specific primers to carry out amplicon targeted enrichment on the connection jointer product; finally, performing PCR amplification and enrichment. The single molecule specific primer designed by the invention obviously improves the number of available effective templates, and the carried adjustment sequence can obviously reduce the probability of forming primer dimers among specific primers, and the utilization rate of templates can be also obviously improved. The ultra-low frequency mutation nucleic acid fragment detection method of the invention uses one-sided single primer amplicon enrichment method and molecular label technology, improves the utilization rate of effective templates, reduces noise sources, reduces false positives, improves detection sensitivity and specificity, and is especially suitable for detection of trace ultra-low frequency mutation samples. At the same time, the invention can also carry out high-efficiency library construction.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to an ultra-low frequency mutation nucleic acid fragment detection method, a mutation nucleic acid fragment library construction method, a primer design method and reagents. Background technique [0002] The detection of somatic mutations by means of Next Generation Sequencing (Next Generation Sequencing) plays an important role in tumor research and tumor diagnosis. However, when applied to the detection of rare tumor mutation genes, the identification of tumor mutation drug resistance, and the evaluation of treatment response effects, the mutation frequency needs to be detected at 0.1% or lower. Since the standard library construction process uses DNA polymerase method to introduce background noise, ultra-low frequency mutation signals will be masked by background noise and difficult to distinguish. The main noise sources can be subdivided into thr...

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Problems solved by technology

[0003] The traditional amplicon enrichment technology is based on double-sided enrichment, which has the problem of cfDNA template loss, and trace amounts of ctDNA slip away

The unilateral enrichment technology can effectively solve the above problems. At present, there are two main unilateral enrichment techniques, one is unilateral enrichment-chain forming (back-to-back primer) mode; the other is unilateral enrichment-nesting PCR primer (nested PCR primer) mode; while the single-test enrichment-strand looping mode has no single-molecule labeling technology for noise reduction, and the single-test enrichment-nested PCR primer mode uses double primers to cause the 3' end of the primer to be detected The loci must be kept at a distance, and there will also be a small amount of template loss, so both seriously affect the detection limit

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