基于连接酶-琼脂糖凝胶电泳的单核苷酸多态性快速检测方法

A rapid detection method for single nucleotide polymorphisms based on ligase-agarose gel electrophoresis

目的研究一种简便、快速、灵敏的单核苷酸多态性（SNP）分型方法,使其能够在简单实验条件下进行常规的临床检测。方法基于连接酶-琼脂糖凝胶电泳技术,设计突变位点的检测探针,通过对检测探针的连接、纯化和通用扩增反应,根据琼脂糖凝胶电泳条带的出现情况判定检测位点的突变类型。以表皮生长因子受体（EGFR）基因的3个SNP突变位点EGFR,c.2573T〉G（L858R）,EGFR,c.2582T〉A（L861Q）和EGFR,c.2155G〉T（G719C）为检测对象,分别对质粒模板和肺癌血浆循环DNA样本进行了检测。结果建立方法操作简便,具有较高的特异性和灵敏度。经过20-30个循环的PCR扩增,能够根据扩增条带的有无清晰判断检测位点的基因型。在对不均一样本中混合等位基因检测时,能够最低检测出2.5%的突变型等位基因。本方法和直接测序法分别能够从62例肺癌样本检测出6例和2例L858R位点杂合子突变。结论 SNP突变检测方法适合于在简单实验条件下对不均一样本进行常规突变检测。

Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the appeared bands in the agarose gel electrophoresis（AGE）.With the 3SNP sites EGFR,c.2573T〉G（L858R）,EGFR,c.2582T〉A（L861Q）and EGFR,c.2155G〉T（G719C）in epidermal growth factor receptor（EGFR）gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6cases and 2cases of heterozygotes mutation in the SNP site of L858 Ramong 62samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condition.