Long-range PCR (Polymerase Chain Reaction) method and kit for detecting polymorphism variation of CYP2D6 gene

Abstract

The invention provides a long-range PCR (Polymerase Chain Reaction) method and kit for detecting polymorphism variation of a CYP2D6 (Cytochrome Phosphate 2D6) gene. The method comprises the following steps: step 1, genome DNA extraction; step 2, specific amplification of the CYP2D6 gene by an LR-PCR method; step 3, purification of an amplification product; and step 4, construction of a product library. Compared with multiple PCR detection, the detection method does not need to design a plurality of pairs of special primers, reduces the amplification of non-specific bands in the PCR process, and effectively enriches the polymorphic variation bands. Under the primer amplification condition, the band of the amplified result is clear and single. Compared with a customized chip hybridization capture technology, Long-range PCR can obviously shorten the time and greatly reduce the cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Long-range PCR method, primer set and kit for detecting polymorphic variation of CYP2D6 gene. Background technique [0002] The CYP2D6 metabolizing enzyme is a member of the cytochrome P450 family, a monooxygenase that catalyzes many reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids. Localized to the endoplasmic reticulum, this protein metabolizes up to 25% of commonly used drugs. Its substrates include antidepressants, antipsychotics, analgesics and antitoxics, beta-adrenergic blockers, antiarrhythmics and antiemetics. CYP2D6 is the only enzyme that cannot be induced in all cytochrome P450 gene families involved in drug metabolism. This enzyme has a wide range of polymorphisms and this polymorphism has an important impact on the drug metabolism function of the enzyme. The polymorphism of CYP2D6 and the difference in individual activit...

AI Technical Summary

Problems solved by technology

The high cost of liquid phase hybridization chip is not suitable for popularization and application

And there are highly homologous regions on the genome in some regions of the gene. When primers or probes are designed for detection of sites in this part of the region, it is easy to be interfered by the homologous regions of the genome, thus affecting the judgment and interpretation of the results.

Moreover, most of the testing products currently have higher requirements for the initial DNA amount of the sample to be tested.

[0006] Chinese patent 201611107790.9 discloses a nucleic acid, kit and method for rapid detection of CYP2D6 gene polymorphism, specifically for the detection of site polymorphisms of CYP2D6*10 gene C100T and G4268C, CYP2D6*14 gene G1758A, However, the entire gene sequence has not been amplified and detected, so it is impossible to more comprehensively and accurately assess the risk of mutation

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