Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Electrophoresis. 2010 Jun;31(11):1790-5. doi: 10.1002/elps.200900664.
The recent discovery of post-transcriptional regulation by microRNAs (miRNAs) drew our attention to SNPs of putative miRNA target sites in candidate genes of depression-related psychiatric disorders. The P2RX7 (purinergic receptor P2X, ligand-gated ion channel, 7) gene has been suggested as a candidate for major depressive and bipolar disorder, because of repeated associations with the rs2230912 (Gln460Arg) polymorphism. As this polymorphism is located at the end of the coding region, we considered a possible linkage with SNP(s) in putative miRNA target sites of the 3' untranslated region. Based on our in silico search, the rs1653625 fulfilled this criterion. This SNP, however, is surrounded with polycytosine and polyadenine tracts, which hindered its analysis until now. In this study, we describe a readily applicable genotyping method for rs1653625 by applying a primer that introduces mismatched nucleotides to create a restriction enzyme cleavage site. The resulting allele-specific products with 19 base pair difference were separated by both traditional horizontal agarose gel electrophoresis and multicapillary gel electrophoresis. The developed genotyping method was applied in our depression-related association study.
最近发现 microRNAs（miRNAs）的转录后调控引起了我们对抑郁症相关精神障碍候选基因中假定 miRNA 靶位点 SNPs 的关注。P2RX7（嘌呤能受体 P2X，配体门控离子通道，7）基因被认为是重度抑郁症和双相情感障碍的候选基因，因为它与 rs2230912（Gln460Arg）多态性的反复关联。由于该多态性位于编码区的末端，我们考虑了 3'非翻译区假定 miRNA 靶位点中的 SNP（s）与它之间可能存在的连锁关系。基于我们的计算机搜索，rs1653625 符合这一标准。然而，这个 SNP 周围环绕着多聚胞嘧啶和多聚腺苷酸，这一直阻碍着对其进行分析。在这项研究中，我们通过应用引入错配核苷酸以创建限制性内切酶切割位点的引物，描述了一种易于应用的 rs1653625 基因分型方法。通过 19 个碱基差异产生的等位基因特异性产物通过传统的水平琼脂糖凝胶电泳和多毛细管凝胶电泳进行分离。开发的基因分型方法应用于我们的抑郁症相关关联研究中。
