小片段DNA未知SNPs的PCR SSCP检测方法分析(英文)

Analysis of PCR-SSCP Detection for Unknown Single Nucleotide Polymorphisms (SNPs) in Small DNA Fragment

PCR SSCP是对未知单核苷酸多态性 (SNPs)进行检测最简便、最实用的方法之一 .该方法检测的灵敏度易受片段大小的影响 .以大小在 2 0 0bp左右的PCR扩增产物为检测对象 ,对该方法的影响因素进行了分析 .结果表明 ,在 4℃条件下 ,10V cm恒压电泳 10～ 16h ,在上样缓冲液中加入10 %甘油 ,在 12 %～ 15 %的非变性聚丙烯酰胺 (arc∶bis =2 9∶1)中加入 5 %甘油可改善分辨效果 ,可作为进行小片段DNA单个碱基突变的一般检测方法 .采用该方法在猪的肌肉生长抑制素基因(MSTN)中鉴定了 3个SNPs ,在猪的雌激素受体基因 (ESR)和兔的酪氨酸酶基因中各检测到 1处SNP ,说明该方法在小片段DNA的检测上是行之有效的 ,为基因组中未知SNPs的检测提供了一种简便的方法 .

Single nucleotide polymorphisms (SNPs) exist widely in genomes. PCR SSCP is one of the simplest and most practical approaches.The detection efficiency of PCR SSCP for SNPs depends greatly on the size of DNA product. In present study, a practical detection procedure for identifying unknown SNPs in DNA fragment around or less than 200 bp was established. By adding 5% glycerol to 12%～15% non denaturing polyacrylamide gel(arc∶bis=29∶1),10% glycerol to loading buffer and running the gel under the ambient temperature of 4℃ at a constant voltage of 10 V/cm, a reasonably good result can be obtained. This method proved to be effective when used in SNPs detection in several genes.