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.

中文翻译：

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探索的想法：使用基于 PCR 的测定法测定酒精代谢相关基因中的单核苷酸多态性，以了解个体基因型和表型之间的联系

在这里，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类乙醇脱氢酶 1B ( ADH1B ) 和乙醛脱氢酶 2 ( ALDH2 ) 基因中的单核苷酸多态性 (SNP)。在本练习中，可以在相同的 PCR 程序（2 步 × 35 个循环，总共 35 分钟）使用发根裂解物作为模板并行。在 AS-PCR 中，两个基因的 G 或 A 等位基因的目标区域在单个 PCR 管中进行等位基因特异性扩增。在PCR-RFLP分析中，两个基因在单管中同时扩增，然后用限制性酶Msl I和Eam 1104I双酶切部分PCR产物5分钟。每种方法得到的反应产物并排进行电泳，并根据 DNA 带模式确定基因型。通过优化的方案，从模板制备到基因分型的整个过程可在约75分钟内完成。在 PCR 过程中，学生还进行乙醇斑贴测试，以评估他们代谢酒精的能力。本系列实验可以帮助学生了解PCR/SNP分析的原理和应用。通过比较PCR显示的基因型和斑贴测试显示的表型，学生可以更好地了解基因检测的临床价值。