HRM method for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T

Abstract

The invention belongs to the field of molecular biology and medicine, and discloses an HRM analysis technology for detecting genetic polymorphism of CYP3A4*1G and MDR1C1236T. The method includes the following steps: (1) searching DNA sequences of target genes; (2) designing and screening appropriate primer pairs aiming at mutation loci of the target genes; (3) extracting sample DNA, and carrying out gene amplification by using the primers screened in the step (2); (4) analyzing whether a PCR product is obtained through specific amplification by an agarose gel electrophoresis method; and (5) according to a high-resolution dissolution curve method, detecting the mutation loci of the amplified target genes, and selecting an SYTO-9 saturated fluorescent dye. A sequence specific probe is not needed during testing, sequencing is not needed, the test is not limited by mutation base loci and types, genetic information is simply and quickly provided for clinical individualized medication, and a help is provided for rational drug use and clinical drug monitoring.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, and relates to gene detection related to pharmacokinetics. In particular, it relates to an HRM method for detecting gene polymorphisms of CYP3A4*1G and MDR1C1236T. The method of the present invention can complete rapid detection of a large number of samples at one time, and the method is simple, accurate, reliable, time-saving and labor-saving, and provides convenience for clinical individualized rational drug use. Background technique [0002] High Resolution Melting Analysis Technology (High Resolution Melting), referred to as HRM, is a tool for SNP and mutation research jointly developed by the University of Utah and Edward Technology Corporation. It can detect 84% of human SNPs (single nucleotide polymorphisms). ). Based on the different physical properties of nucleic acid molecules affecting the melting temperature, HRM detects the combination of saturated fluorescent dyes and PC...

AI Technical Summary

Problems solved by technology

The RFLP method combines PCR with restriction enzyme digestion, because of its cumbersome experimental operations, long detection cycle, incomplete first round of enzyme digestion leading to false positive results, and can only be used for the judgment of known SNPs, there is no restriction site It is only suitable for the detection of a small number of samples due to factors such as the inability to detect

The denaturing gradient electrophoresis method has a commercial electrophoresis device, which is suitable for the detection and screening of large samples, but because the method uses temperature and gradient gel mobility for detection, gel electrophoresis is required, and the secondary structure of the DNA chain is likely to cause artificial false phases , leading to biased results

In the DNA direct sequencing method, the PCR products are sequenced after electrophoresis on a fully automatic sequencer. The method is advanced, but the workload is heavy, the time is long, and the price is expensive. It is not suitable for the detection of large samples.

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