SENSITIVE DETECTION OF P53 GENE-MUTATIONS BY A MUTANT ENRICHED PCR-SSCP TECHNIQUE

For the rapid and sensitive detection of p53 'hot spot' mutations, we combined polymerase chain reaction based single-strand conformational polymorphism (PCR-SSCP) analysis with sequence specific-clamping by pe ptide nucleic acids (PNAs) in a one-step reaction tube protocol, For t his purpose, we designed two PNA molecules comprising aa 246-250 of ex on 7 and aa 270-275 of exon 8, respectively, to suppress the amplifica tion of wild-type p53 allelic variants during PCR amplification, Using this method in a survey of 20 brush cytology samples from lung cancer patients, we were able to detect five p53 point mutations occurring i n codons 248, 249 and 273 which could not be retrieved by conventional PCR-SSCP. Thus, allelic suppression by PNA molecules opens a way to l argely improve the sensitivity of existing PCR-SSCP protocols (similar to 10-50-fold) and could be useful in the detection of 'hot spot' onc ogene lesions in histological samples containing only a small number o f cancer cells.