A low-abundance gene mutation enrichment method based on the removal of wild-type sequences

Abstract

The invention discloses a low-abundance gene mutation enrichment method based on the removal of wild-type sequences. The method of the invention processes the sample DNA used as a DNA template before PCR amplification, so that the 3' end is blocked and contains mutations. The nucleotide single strand at the site is used as a hybrid strand to hybridize with the wild-type and mutant sequences, and the double-strand-specific nuclease can specifically remove the homologous double-stranded DNA that is completely complementary to the wild-type sequence formed by the hybrid strand, However, the non-completely complementary heteroduplex DNA formed by the hybrid strand and the mutant sequence cannot be removed. The removed product is amplified by PCR, and the 3' end of a primer used in PCR amplification is located at the mutation site and is blocked. Under the action of a polymerase with 3'-5' proofreading activity, the wild-type sequence cannot However, the mutant sequence can be amplified exponentially, thereby effectively enriching the low-abundance gene mutant sequence and improving the enrichment sensitivity and accuracy. At the same time, the method of the present invention can also simultaneously enrich a variety of mutant sequences so as to improve the amplification efficiency. increase efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a low-abundance gene mutation enrichment method based on removing wild-type sequences. Background technique [0002] The completion of the Human Genome Project has promoted the rapid development of gene mutation research. In recent years, the proposal of the "Precision Medicine" program has further made the study of gene mutations and their detection methods a hot spot and basis for biological research and clinical precision medicine. With the continuous development of detection technology, researchers have found that most gene mutations exhibit low abundance (content ranges from 0 to 100%), and these low abundance gene mutations have important research significance in the fields of medicine and biology. Emerging new methods and technologies provide reliable prospects for the enrichment of low-abundance gene mutations, most of which are based on the principle of PCR technology. PCR...

AI Technical Summary

Problems solved by technology

Generally, high-throughput sequencing cannot detect DNA mutations with an abundance of less than ~2%. The method of removing a large number of wild-type DNA sequences can effectively enrich low-abundance mutant sequences in order to improve the detection limit of sequencing.

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