PCR-SSCP分析方法的优化

被引：6

作者：

王娜

连易水

刘砚星

机构：

[1] 河北医科大学基础医学院药理学教研室

来源：

现代生物医学进展 | 2008年 / 10期

关键词：

SSCP; 突变; 聚丙烯酰胺凝胶; 银染;

D O I：

10.13241/j.cnki.pmb.2008.10.004

中图分类号：

Q503 [生物化学技术];

学科分类号：

071010 ; 081704 ;

摘要：

目的:优化PCR-SSCP分析方法。方法:选择野生型Kir2.3质粒和突变型Kir2.3(I2123L)质粒为样本,其PCR结果的鉴定采用2%的琼脂糖凝胶EB显色,SSCP分析采用12%的非变性聚丙烯酰胺凝胶DNA银染显色。PCR产物变性比较了三种方法:即碱变性、SDS变性、直接变性。聚丙烯酰胺凝胶根据丙烯酰胺和甲叉双丙烯酰胺及甘油的比例设计了六个实验组,每组采用两种条件电泳,即:30mA恒流快速电泳和100V恒流过夜电泳。结果:选择直接变性的PCR产物作为变性上样液,并确定了三种凝胶配及该条件下适用的电泳条件。即:丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含1%甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含5%甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为29比1,凝胶中不含甘油,4℃,500V高压电泳3min接着100V恒流过夜电泳12h。结论:条件优化的PCR-SSCP是一种筛查基因突变简便、有效的实验方法。

引用

页码：1841 / 1844+1825 +1825

页数：5

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