基于荧光定量PCR技术的ADH1B基因多态性分型教学实验设计

Experimental design of ADH1B gene polymorphism typing teaching based on fluorescence quantitative PCR technology

该实验设计旨在指导学生使用不同的PCR衍生技术进行基因分型检测。实验设计了高分辨率熔解曲线分析(PCR-HRM)、等位基因特异性PCR(AS-PCR)和TaqMan探针三种PCR方法,检测了ADH1B rs1229984位点的基因多态性。三种PCR检测结果与Sanger测序结果一致。通过该实验,学生能够学习基因多态性的分子机制和相关检测技术,提高动手实操能力,还能学习利用软件和算法设计复杂PCR引物和探针方法及其在临床诊断中的应用。

[Objective]Genetic polymorphisms determine individual genetic differences and disease susceptibility and affect drug metabolism and transport.Genetic polymorphism testing has significant application value in various fields,such as medical research,disease diagnosis,prevention,and health management.Fluorescence quantitative polymerase chain reaction(qPCR)instruments are highly sensitive and accurate molecular biology detection tools widely used in basic medical research and clinical diagnosis.Thus,medical students need to learn the fluorescence qPCR technique to improve their research capabilities and disease diagnosis levels and respond to public health events.This experiment designs three methods,namely high-resolution melting curve(HRM),allele-specific PCR(AS-PCR),and TaqMan probe,to genotype the ethanol dehydrogenase 1B(ADH1B)rs1229984 locus,aiming to guide students in using different PCR-derived techniques for genetic polymorphism testing.[Methods]The ADH1B gene sequence and the homologous genes ADH1C and ADH1A with high homology were downloaded from the GenBank database.The SnapGene software for gene alignment was used to locate the rs1229984 locus.Amplification primers for HRM-PCR,AS-PCR,and direct sequencing were designed using software such as SNPway,Primer,and Beacon Designer and algorithms such as NUPACK and MetLab.Commercial TaqMan probe primer pairs were ordered.Oral epithelial cells were successively collected from 48 volunteers,DNA was extracted using a rapid extraction method,and DNA content was measured using a spectrophotometer.PCR amplification systems were configured for the HRM,AS,and TaqMan methods,amplification reactions were performed on the corresponding PCR instruments,and the corresponding software was used for result analysis after the reaction ended.At the same time,the PCR products were detected using Sanger sequencing to identify the genotype of ADH1B rs 1229984.[Results]The HRM,AS,and TaqMan methods can all accurately identify the polymorphism of the ADH1B gene,and their genotyping results are consistent with the results of the direct and Sanger sequencing methods.Each of the three PCR methods has its advantages and disadvantages:The TaqMan method has strong sensitivity and specificity but requires designing a pair of primers in addition to a pair of locus-specific probes,which is costly.The HRM method is the simplest to operate and the lowest in cost,but it requires high-quality DNA.The AS method has strong specificity but requires high primer design standards and involves PCR amplification reactions with two upstream primers and one downstream primer,comparing theΔCt values of the two for judgment,which is relatively complex.[Conclusions]By establishing the HRM,AS,and TaqMan methods,the polymorphism of the ADH1B gene rs122998 locus in 48 volunteers was successfully genotyped.The accuracy of the experimental results confirmed the reliability of fluorescence qPCR technology in the analysis of genetic polymorphisms.Through experimental teaching,students can be taught to apply what they have learned to detect other genetic polymorphisms.When choosing a detection method,several factors,such as the purpose of the experiment,number of samples,cost,and ease of operation,need to be considered comprehensively.