Method for amplifying nucleic acid and application thereof

Abstract

The invention discloses a method for amplifying nucleic acid and application thereof. The method for amplifying nucleic acid comprises the step of performing primer extension on an extension reaction template by using an extension primer, thereby acquiring an extension product, namely, the amplified nucleic acid. The primer extension is composed of n circular extension reactions; n is a natural number from 5 to 25; each circular extension reaction in n circular extension reactions is composed of two steps; the first step of each circular extension reaction in n circular extension reactions is performed under the conditions of the reaction temperature at 94 DEG C and the reaction time is 30 seconds; the reaction temperature and the reaction time in the second step of n circular extension reactions are both increasing; the increasing rate of the reaction temperature in the second step is at 0.5 DEG C / circle; the increasing rate of the reaction time in the second step is at 20-30 seconds / circle. According to the method for amplifying nucleic acid provided by the invention, the amplification efficiency of the nucleic acid extension reaction template can be improved.

Description

technical field [0001] The invention relates to a nucleic acid amplification method and its application in the field of biotechnology. Background technique [0002] EGFR is the epidermal growth factor receptor, and EGFR mutations occur in 57% of glioblastoma patients. Moreover, there are many types of EGFR mutations, involving various mutation modes such as gene amplification, gene rearrangement, insertion-deletion, and point mutation. Epidermal growth factor receptor variant type III (EGFRvIII) mutations are common in glioblastoma, and about 50% of EGFR amplification mutant gliomas are accompanied by EGFRvIII mutations. At present, the main detection methods for EGFRvⅢ mutations are 1) real-time quantitative PCR detection at the RNA level; 2) next-generation sequencing to detect somatic mutations. Fluorescent quantitative PCR detection at the RNA level cannot identify mutations at the genomic DNA level, and RNA is easier to degrade than DNA, and the detection sensitivity ...

AI Technical Summary

Problems solved by technology

The long-Range PCR method used by Lori Frederick can detect EGFRvⅢ mutations at the level of genomic DNA. The specific scheme is as follows: two sets of upstream primers are designed, and the distance between these two sets of adjacent primers is about 5kp, and each set of primers is respectively Mix together, as upstream mixed primers, and perform PCR reactions with downstream primers respectively, but this method is not sensitive and there are many non-specific amplification phenomena

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