Atha D H, Wenz H M, Morehead H, Tian J, O'Connell C D
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Electrophoresis. 1998 Feb;19(2):172-9. doi: 10.1002/elps.1150190207.
We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrophoresis (CE-SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG-SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using the Perkin Elmer ABI PRISM 310 Genetic Analyzer with GeneScan Software and the Beckman P/ACE 5510 CE equipped for laser-induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild-type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrument. For mutations measured at ambient temperature (25 degrees C), characteristic shifts in direction and magnitude were observed in the migration times of both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitude and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature-specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE-SSCP. In addition, by using CE, discrete intra-strand isoforms could be easily observed at different temperatures. The combination of mutation-specific temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutations.
我们使用毛细管电泳单链构象多态性分析（CE - SSCP）对五个p53单点突变进行了分析，并将这些测量结果与通过平板凝胶电泳（SG - SSCP）获得的测量结果进行了比较。使用PCR引物扩增特定外显子以进行突变检测。5'引物用FAM（5 - 羧基荧光素）标记，3'引物用JOE（2'，7'-二甲氧基 - 4'，5'-二氯 - 6 - 羧基荧光素）标记。使用配备GeneScan软件的Perkin Elmer ABI PRISM 310遗传分析仪和配备激光诱导荧光检测的Beckman P/ACE 5510 CE进行CE - SSCP分析。尽管通过SG或CE分析都能成功检测到p53突变相对于相应野生型链的迁移时间变化，但毛细管电泳的单次电泳运行时间快约十倍且自动化程度更高。CE - SSCP测量在原型仪器上于10至60摄氏度的温度范围内进行。对于在室温（25摄氏度）下测量的突变，相对于野生型，所有突变的两条链的迁移时间在方向和幅度上都观察到了特征性变化。这证明了室温下CE解析这些突变的能力。然而，迁移时间变化的幅度和方向因每个突变而异，并且每个突变都呈现出特定于温度的图谱。这表明在通过CE - SSCP解析单点突变时，扩展的温度控制将是一个重要优势。此外，通过使用CE，可以在不同温度下轻松观察到离散的链内异构体。CE - SSCP的突变特异性温度图谱分析和异构体分析相结合，应该有助于诊断界检测基因突变。
