Multiplex-PCR three-round amplification method

Abstract

The invention relates to a multiplex-PCR three-round amplification method, a primer and a kit thereof and application of the method in establishment of next-generation sequencing platform libraries. In the first two rounds of a PCR, a specific primer with the low concentration is consumed as much as possible, so that the differences of different amplicons are reduced and the homogeneity of multiplex-PCR products is improved; when the third round of the PCR is performed, adapter primers carrying different bar code sequences are added to mark different templates, the different adapter primers carry same universal amplification primers, therefore, it is guaranteed that the amplification efficiency of the different templates is consistent, and the differences of different template amplification products are reduced. Accordingly, not only is the problem that the homogeneity of ordinary multiplex-PCR amplification is poor solved, but also establishment of the sequencing libraries can be quickly and conveniently completed.

Description

technical field [0001] The present invention relates to the field of nucleic acid detection, in particular to a technique for enriching the DNA sequences of multiple sample target segments through three rounds of multiplex PCR, and constructing uniform enriched fragments into libraries suitable for next-generation sequencing platforms. Background technique [0002] Nucleic acid is the basis of all genetic material. Since the genetic code was cracked, the study of the relationship between genotype and phenotype has been the tireless pursuit of geneticists. Researchers hope to clarify the relationship between genetic mutations and biological functions at the genome level, hoping to make new breakthroughs in genetic diseases, evolutionary origins, and disease localization. [0003] In the past thirty years, nucleic acid sequencing still relies on capillary electrophoresis based on sanger sequencing; and in the past decade, the rapid development of next-generation sequencing tec...

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a method for establishing a uniform library suitable for next-generation sequencing platforms by three rounds of multiplex PCR, so as to overcome the problems of inhomogeneity of different samples and sites in the existing multiplex PCR technology, as well as the operation process of the detection scheme Complex, poorly specific defects

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