van de Locht L T, Kuypers A W, Verbruggen B W, Linssen P C, Nováková I R, Mensink E J
Department of Hematology, University Hospital Nijmegen, The Netherlands.
Thromb Haemost. 1995 Nov;74(5):1276-9.
Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.
最近，凝血因子V基因中的一个点突变（G1691A）被证明可导致对活化蛋白C切割的抗性。该突变与血栓形成风险增加相关，是迄今为止血栓形成倾向最常见的遗传原因。目前在DNA水平研究单碱基对突变的技术是基于聚合酶链反应，随后进行限制性酶切或Southern印迹以及等位基因特异性探针检测的分析方法。我们在此描述的方法包括一个单一的聚合酶链反应，其中在一个反应中使用两个专门设计的等位基因特异性引物和两个共有引物，以区分纯合正常、杂合和纯合突变个体。使用毛细管电泳和在线紫外监测分析扩增产物。等位基因特异性扩增方案及后续的毛细管电泳分析（ASAP-CE）是一种方便、快速、自动化且高度可重复的方法，可用于常规实验室环境。
