Abstract：
Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele-specific (AS) PCR and a PCR-restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2-step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS-PCR, the target regions of the G- or A-alleles of both genes are allele-specifically amplified in a single PCR tube. In the PCR-RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double-digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.
摘要：
这里,我们提出了一项实验室练习,以快速确定参与酒精代谢的人酒精脱氢酶1B(ADH1B)和醛脱氢酶2(ALDH2)基因中的单核苷酸多态性(SNPs).在这个练习中，基于聚合酶链反应（PCR）的两种不同的基因分型方法，即等位基因特异性（AS）PCR和PCR限制性片段多态性（RFLP）分析，可以在相同的PCR程序下进行(2步×35个循环，总共35分钟）平行使用发根裂解物作为模板。在AS-PCR中,例如,两个基因的G-或A-等位基因的靶区域在单个PCR管中进行等位基因特异性扩增。在PCR-RFLP分析中,这两个基因在一个试管中同时扩增，然后用限制酶MslI和Eam1104I对一部分PCR产物进行5分钟的双重消化。将每种方法得到的反应产物并排电泳,从DNA条带模式确定基因型。有了优化的协议，从模板制备到基因分型的整个过程可以在约75分钟内完成。在PCR过程中,学生还进行了乙醇斑贴试验，以估计他们代谢酒精的能力。这一系列实验可以帮助学生学习PCR/SNP分析的原理和应用。通过比较PCR揭示的基因型和斑贴试验揭示的表型，学生可以更好地了解基因检测的临床价值。
