Arakawa H, Tsuji A, Maeda M, Kamahori M, Kambara H
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
J Pharm Biomed Anal. 1997 Jun;15(9-10):1537-44. doi: 10.1016/s0731-7085(96)01988-7.
Detection of point mutations in genomic DNA is important for diagnosis of inherited characteristics and genetic diseases. A point mutation in a specific region of DNA amplified by polymerase chain reaction (PCR) can be detected with single-strand conformation polymorphism (SSCP) analysis. Analysis of SSCP by laser-induced fluorescence capillary electrophoresis in entangled polymer solution (CE-LIF) has been developed in the present paper. K-ras genes including seven mutations were amplified with primer labeled with Texas Red at its 5' end. The labeled PCR products were dissociated to single strands by heating and separated with capillary gel electrophoresis and He-Ne laser-excited fluorescence detection. Our results suggest that all fragments having normal (Gly) and mutated (Ala, Arg, Cys, Ser, Val, Asp) sequences at codon 12 can be distinguished. Analysis of SSCPs with CE-LIF is well suited for clinical analysis of SSCPs because of its high sensitivity, resolution, reproducibility and speed.
检测基因组DNA中的点突变对于遗传性特征和遗传疾病的诊断非常重要。通过聚合酶链反应（PCR）扩增的DNA特定区域中的点突变可以用单链构象多态性（SSCP）分析来检测。本文开发了在缠结聚合物溶液中通过激光诱导荧光毛细管电泳分析SSCP（CE-LIF）的方法。用在其5'端标记有德州红的引物扩增包含七个突变的K-ras基因。标记的PCR产物通过加热解离为单链，并通过毛细管凝胶电泳和氦氖激光激发荧光检测进行分离。我们的结果表明，所有在密码子12处具有正常（甘氨酸）和突变（丙氨酸、精氨酸、半胱氨酸、丝氨酸、缬氨酸、天冬氨酸）序列的片段都可以区分。由于其高灵敏度、分辨率、重现性和速度，用CE-LIF分析SSCP非常适合于SSCP的临床分析。
