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 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.
Atha
D H
DH
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. donald.atha@nist.gov
Wenz
H M
HM
Morehead
H
H
Tian
J
J
O'Connell
C D
CD
D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016159:Tumor Suppressor Protein p53