DETECTION OF P53 POINT MUTATIONS BY SINGLE-STRAND CONFORMATION POLYMORPHISM - ANALYSIS BY CAPILLARY ELECTROPHORESIS

We have analyzed five p53 single point mutations by single strand conf ormation polymorphism using capillary electrophoresis (CE-SSCP) and ha ve compared these measurements to measurements obtained by slab gel el ectrophoresis (SG-SSCP). PCR primers were used for amplification of sp ecific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE 7'-dimetho xy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using t he Perkin Elmer ABI PRISM(TM) 310 Genetic Analyzer with GeneScan(TM) S oftware and the Beckman P/ACE(TM) 5510 CE equipped for laser-induced f luorescence 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 ele ctrophoresis run times were about tenfold faster and more automated wi th capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrum ent. For mutations measured at ambient temperature (25 degrees C), cha racteristic shifts in direction and magnitude were observed in the mig ration times of both strands of all mutations relative to the wild typ e. This demonstrated the ability of CE at ambient temperature to resol ve these mutations. However, the magnitute 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 mutat ion. This demonstrated that extended temperature control will be an im portant advantage in resolving single point mutations by CE-SSCP. In a ddition, by using CE, discrete intra-strand isoforms could be easily o bserved at different temperatures. The combination of mutation-specifi c temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutati ons.