Method of detecting single base mutation and application

Abstract

The invention discloses a method of detecting single base mutation and application. The method includes the steps that primers are designed targeted to a sample to be detected and a target segment for reference, and PCR is subjected to amplification; amplification products are subjected to balanced mixing, high-temperature denaturation is conducted, then annealing is conducted, heteroduplex DNA is obtained, then CEL I enzyme digestion is conducted, an enzyme-digested product is subjected to electrophoretic determination, and a single-base mutation sample is determined; two segments subjected to CEL I enzyme digestion are recycled, and each segment is connected with a connector provided with an A, or T, or G or C sticky tail end; one of upstream primers and downstream primers for amplification of the target segment and one of forward and reverse primers designed based on the connector sequence form two pairs of primers, each connection product serves as a template, and PCR amplification is conducted; electrophoretic detection is conducted. By means of the method for detecting single base mutation, known single base mutation can be detected, unknown single base mutation can be further screened, the position of mutation can be determined, the type of mutation can be further determined, sensitivity is high, repeatability is good, and the economic property is achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for detecting a single base mutation. Background technique [0002] Single nucleotide variation (point mutation) refers to the change of one nucleotide at a specific position in DNA, which widely exists in the coding of genetic information of organisms. When the allele frequency of rare occurrence is greater than or equal to 1%, It is called single nucleotide polymorphism (singlenucleotidepolymorphism, SNP). The study of point mutations helps to understand the genetic characteristics of higher organisms, explain the phenotypic differences of individuals, reveal the normal functions of genes, information, proteins, the causes of variation, and the response mechanism of different individuals to environmental changes. Functional genomics Research and establishment of molecular markers are of great significance. [0003] With the development of genome sequencin...

AI Technical Summary

Problems solved by technology

Although with the improvement of sequencing automation and the reduction of sequencing costs, direct sequencing is more and more used in SNP detection, but repeated sequencing and confirmation are required to distinguish heterozygotes from sequence errors, and the cost of sequencing is still very high and time consuming

[0011] As can be seen from the above, with the continuous emergence of new technologies, new materials and equipment, point mutation detection technology has been developed rapidly, but most of them cannot determine the specific mutation base, but can determine the method of mutation base (such as Sequencing method) requires specialized analytical instruments, which are not only expensive, but also complicated to operate and highly professional, and are subject to certain restrictions in application.

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