Method for detecting the polymorphism of ADH2 genes

Abstract

The invention pertains to the field of biotechnology and relates to a method for detecting the polymorphism of ADH2 genes. Based on the feature analysis of ADH2 genes and relevant polymorphic base sequences thereof, primers are designed according to the principle that mismatched basic groups create enzyme cutting sites; one of the primers is designed according to the neighboring sequence of a mutant site; mismatched basic groups are introduced to cause the end of a primer 3' and a mutant of the monobasic mutation to form an enzyme cutting site after the amplification of polymerase chain reaction (PCR); and then, incision enzymes are used for enzyme cutting identification. By experimental verification, the design of primers is reasonable and the amplification of PCR is successful, the polymorphism of amplified DNA fragments can be identified by incision enzymes including HhaI, HinP1I, BstUI and isoenzymes thereof. The electrophotogram of BstUI after enzyme cutting has clear straps and is easy to identify. The used incision enzymes are low in price and easy to obtain. According to sequencing verification, the features of sequences with different genotypes are consistent with the expected results. According to the results of verification on population-based samples, the method is stable and reliable.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a detection method of ADH2 gene polymorphism (rs1229984). Background technique [0002] Studies have reported that after ethanol is ingested into the human body, it is quickly digested by the mouth, esophagus, stomach, and intestines, among which the liver is the most important organ for its metabolism, and 90%-98% of alcohol is metabolized in the liver. Under normal circumstances, ethanol at low blood levels is mainly metabolized by the ADH ethanol oxidation system, while high-concentration ethanol is mainly metabolized by the microsomal ethanol oxidation system during long-term drinking [1] . There are gene polymorphisms in ADH2 and ADH3 in the ADH system, and they express different enzyme activities, which in turn affect the susceptibility of the body to ethanol. Among them, ADH2 is the most important, so the sensitivity of alcohol drinkers with different genotypes to ethan...

AI Technical Summary

Problems solved by technology

All there are following deficiencies in the prior art: the required instruments and reagents of techniques such as Taqman are expensive, and are usually used by large research institutions and companies; The steps of acrylamide gel electrophoresis and silver staining are cumbersome; in the application of the PCR-CTPP method, an artificial mismatched base is introduced into the 3' end region of the primer to improve the specificity of the extension reaction, and the specificity of the multiplex PCR reaction is uniform. Can lead to non-specific bands and system instability

[0003] The classic PCR-RFLP method is the most commonly used in gene polymorphism detection because of its simple operation, rapidity, and accurate end point judgment. This method was often used in the early ADH2 gene polymorphism detection, but the source of MaeШ required for it is scarce and expensive , which to some extent limits the development of related research

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