Identification of known p53 point mutations by capillary electrophoresis using unique mobility profiles in a blinded study.
This study is part of an ongoing project at the National Institute of Standards and Technology (NIST) that generates a panel of DNA clones containing the most common mutations found in the human p53 tumor suppressor gene. This panel will be made available as a reference source for evaluation and testing for p53 mutations. Single strand conformation polymorphism (SSCP) analysis has found widespread acceptance as a tool for simply and rapidly screening for mutations, albeit with a detection rate that can be below 100%. We have begun to analyze mutations found in exon 7 of the p53 gene by SSCP using laser induced fluorescence capillary electrophoresis (LIF-CE). PCR fragments, containing single point mutations, were amplified from genomic DNA isolated from cell lines using primers labeled with two different fluorophores. This dual labeling approach allowed better traceability of mobility shifts as a function of the experimental conditions. While analyzing the clones H596, Colo320, Namalwa and wild type (reference samples) at different temperatures, ranging from 25 to 45 degrees C, it was observed that each mutation responded in a unique way to changes in temperature both in magnitude and direction of shifts relative to the wild type sample. In a blinded study, ten p53 exon 7 samples were matched automatically, using ABI PRISM Genotyper software, against the four reference samples. From these 10 samples, six were correctly identified as containing one of the reference mutations, two corresponded to wild type, and two were correctly identified as non-reference mutations. This approach should prove helpful in the rapid screening of target sequences that are known to bear a limited number of mutations.
PE Applied Biosystems, Foster City, CA 94404, USA. wenzmh@perkin-elmer.com
H M
HM
Wenz
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S
Ramachandra
C D
CD
O'Connell
D H
DH
Atha
D001331:Automation; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016158:Genes, p53; D006801:Humans; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D016037:Single-Blind Method; D012984:Software; D013696:Temperature; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D005452:Fluoresceins; D005456:Fluorescent Dyes; C045132:4-carboxyfluorescein; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein