Screening of Point Mutations on p53 Gene in Primary Lung Cancer using Single-Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction

Abstract

Single-strand conformation polymorphism (SSCP) analysis was applied to detect mutations of p53 gene in 32 patients with primary lung cancer. cDNA was synthesized from total RNA of primary or metastatic tissues of lung cancer using oligo (dT) primer and reverse transcriptase.³⁵S-labelled partial cDNA with various lengths (200-400bp) were amplified from evolutionary conserved regions of p53 gene by polymerase chain reaction (PCR) using pairs of p53 specific primers and [α-³⁵S] dCTP. After denaturing by formamide, the amplified products were electrophorezed on nondenaturing polyacrylamide gel, and electrophoretic mobilities were compared. Among 32 specimens, 23 were found to have different electrophoretic mobilities compared with normal specimens. Ten out of 23 cases were subjected to further analysis of nucleotide sequencing. Point mutations were found at amino acid residues 151 (Pro→Ser), 234 (Tyr→His, 2 cases), 245 (Gly→Ala), 248 (Arg→Leu), 249 (Arg→Met), 266 (Gly→Arg), 273 (Arg→Leu, 2 cases) and 283 (Arg→Pro). In SSCP analysis, no heterozygosity of abnormal or normal bands was found in any case, suggesting that combination of point mutation and deletion of another allele is the main mechanism of loss of p53 function in primary lung cancer. When SSCP analysis was performed using PCR fragments of 200-400bp length, detectability of p53 point mutations by SSCP analysis seemed to depend on neither length of the PCR fragments nor position of point mutations on each PCR fragment. PCRSSCP analysis is useful for screening of a large number of clinical specimens in the study of p53 gene analysis.