Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fuorescence-based single strand conformation polymorphi sm (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer f or unique color defection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell li nes with reported mutations in p53 exons 5-8 were analyzed by SSCP using va rious gel conditions. The sensitivity for mutation detection was 95% for al l cell lines studied, and no false positives were seen in 10 normal DNA sam ples for all four exons. Experiments mixing known amounts of tumor and norm al DNA showed that mutations were detected even when tumor DNA was mixed wi th 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a u seful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.