Imai Miho, Kisoi Madoka, Sakaguchi Yui, Yamamura Miwako, Kawai Sayuri, Murata Shigenori, Ichikawa Atsushi, Kinoshita Kenji
School of Pharmaceutical Sciences, Mukogawa Women's University.
Institute of Biosciences, Mukogawa Women's University.
Yakugaku Zasshi. 2019;139(8):1111-1119. doi: 10.1248/yakushi.19-00016.
A new single nucleotide polymorphisms (SNP) genotyping method has been developed and validated using biological specimens directly as templates for TaqMan PCR without general DNA extraction and purification procedure from dried saliva samples attached on water-soluble papers. This new method can set up at ease and complete PCR analysis including data interpretation in under two hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost. Specifically, SNP genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) were demonstrated by TaqMan PCR assay using dried saliva samples in the present investigation. In this protocol, by simplifying experimental operations and improving efficiency, omitting and simplifying the time and laborious DNA purification process, it is possible to shorten the experiment time and reduce the risk of human error such as contamination. Furthermore it became possible with great cost reduction. We succeeded in dramatically improving the judgment rate and accuracy of SNP genotyping by the master mix reagent for commercial available real-time TaqMan PCR. Moreover, it becomes possible to stably introduce template DNA into the reaction system, and it will be possible to apply it to copy number variation (CNV) by TaqMan probe method. The SNP analysis process using this optimized water-soluble paper will be applied to gene polymorphism analysis of drug metabolizing enzyme gene CYP, etc., to help efforts to realize personalized medicine.
一种新的单核苷酸多态性（SNP）基因分型方法已经开发并得到验证，该方法直接使用生物样本作为TaqMan PCR的模板，无需从附着在水溶性纸上的干燥唾液样本中进行常规DNA提取和纯化程序。这种新方法可以轻松设置，并在两小时内完成包括数据解读在内的PCR分析，还具有低成本应用于大规模临床研究、诊断和流行病学研究的额外优势。具体而言，在本研究中，通过TaqMan PCR检测，使用干燥唾液样本对酒精代谢相关基因ADH1B（rs1229984）和ALDH2（rs671）进行了SNP基因分型。在该方案中，通过简化实验操作和提高效率，省略并简化了耗时费力的DNA纯化过程，从而有可能缩短实验时间并降低污染等人为误差风险。此外，还实现了大幅降低成本。我们通过市售实时TaqMan PCR的预混试剂成功显著提高了SNP基因分型的判断率和准确性。而且，能够稳定地将模板DNA引入反应体系，并且可以通过TaqMan探针法将其应用于拷贝数变异（CNV）分析。使用这种优化的水溶性纸的SNP分析过程将应用于药物代谢酶基因CYP等的基因多态性分析，以助力实现个性化医疗的努力。
