巢式PCR-RFLP检测rs9263726位点的实验研究

Study on the Detection of rs9263726 Locus by Nested PCR-RFLP

摘要: 目的：rs9263726位点(G>A)之A等位基因是预测别嘌呤醇致严重不良反应风险的遗传标记，本研究旨在建立一种直接使用口腔上皮细胞进行PCR结合FokI酶切检测该位点基因型的方法。方法：自行设计扩增产物包含FokI内对照识别序列的引物，以样本的口腔上皮细胞粗处理物为模板，通过巢式PCR扩增跨越rs9263726位点的靶片段，进而根据PCR产物的FokI酶切图谱判断样本基因型。结果：所检样本均能扩增出预期大小的PCR产物，3种基因型的酶切图谱特征明显，且经测序验证结果一致。结论：初步建立了一种基于巢式PCR-RFLP检测rs9263726位点的改良方法，具有简便、快速、准确、低成本等特点。

Abstract: Objective: The A allele of rs9263726 locus (G>A) is a genetic marker for predicting the risk of serious adverse reactions caused by allopurinol. The purpose of this study is to establish a method to detect this locus using a combination of direct PCR from oral epithelial cells with FokI restriction digestion. Methods: Using innovational self-designed primers, a target fragment spanning the rs9263726 locus and upstream containing the FokI internal control restriction site was amplified by nested PCR from crudely-treated oral epithelial cells, and the genotype of the samples was determined by FokI digestion restriction map of PCR products. Results: The PCR products of expected size could be amplified from all samples, and the digestion patterns of three different genotypes were clearly distinguished, which were consistent with the sequencing results. Conclusion: An improved method based on nested PCR-RFLP to detect rs9263726 locus has been preliminarily established. It is simple, rapid, accurate and at low cost.