四引物扩增受阻突变体系PCR在绵羊线粒体基因组SNP检测中的应用  

Application of Tetra-primer ARMS PCR in Detecting SNP Genotype of Sheep Mitochondrial Genome

作　　者：陈晓勇[1,2] 向海[1] O.H.D.Brahi 敦伟涛[2] 赵兴波[1] 

机构地区：[1]中国农业大学动物科技学院,北京100193 [2]河北省畜牧兽医研究所,保定071000

出　　处：《中国草食动物科学》2013年第6期5-8,共4页China Herbivore Science

基　　金：转基因生物新品种培育重大专项(2013ZX08009-002);河北省农业科技成果转化资金项目(11220139D)

摘　　要：为建立绵羊线粒体基因组SNP的四引物扩增受阻突变体系(tetra-primer amplification refractory mutation system,Tetra-primer ARMS)PCR检测方法,选择小尾寒羊线粒体T1112C和C11606T2个SNP,在等位基因特异PCR基础上,根据错配原理设计4条引物,优化PCR反应体系,采用琼脂糖凝胶电泳检测PCR产物。结果表明:T1112C位点检测中,所有个体均能扩增出569 bp最长片段的外引物产物(阳性对照),扩增出349 bp片段的个体为野生型,扩增出263 bp片段的个体为突变型。C11606T位点检测中,所有个体均能扩增出572 bp最长片段的外引物产物(阳性对照),扩增出408 bp片段的个体为野生型,扩增出213 bp片段的个体为突变型。在每个线粒体多态位点均检测到野生型和突变型2种基因型,没有发现异质性。应用Tetra-primer ARMS PCR方法,经一次PCR即可判定线粒体SNP位点的基因型,该方法快速、简便,可应用于绵羊线粒体SNP分型。The single nucleotide polymorphisms of T1112C and C11606T,as the examples for establishing tetra-primer amplification refractory mutation system polymerase chain reaction（tetra-primer ARMS PCR） in Small-tail Han sheep,were detected by designed four primers according mismatch principle of Allele-specific PCR,optimized PCR system and electrophoresis.The results were as follow：all samples could amplified the longest segments （569 bp and 572 bp） which were the positive contrast in detection of T1112C and C11606T respectively,the samples which visualized 349 bp or 408 bp only after electrophoresis were wild genotypes.Accordingly,if there appeared 263 bp or 213 bp only after electrophoresis,the samples were mutation genotypes.There were two genotypes （wild and mutation genotype） in SNPs of all samples without heterogeneity.Tetra-primer ARMS PCR is a simple,rapid and efficient method in genotyping of SNP from sheep mitogenome by once PCR.

关 键 词：四引物扩增受阻突变体系PCR 绵羊 线粒体 单核苷酸多态性 

分 类 号：S826.2[农业科学—畜牧学]