A highly sensitive detection method for somatic mutations in p53 gene.

A simple and highly sensitive detection method for somatic mutations, which is applicable to diagnostic samples is developed. This method detects somatic mutations that are lower than several percent in abundance. The unique feature with this method is that single-nucleotide primer extension is carried out in the absence of dye terminators that carry respective bases in the wild type gene. Data of mutation sites and bases in the target genes are obtained from databases. Primers of different nucleotide length are designed so that electrophoreses of the single-nucleotide primer extension products allow identification of respective mutations. Based on somatic mutation data in IARC TP53 mutation database, this method was applied to p53 somatic mutations. Results showed that extension reactions in 5 separate tubes allowed detection of mutations in human p53 gene for the most frequent 12 mutations in a nucleotide sequence of 14,049-14,522, which cover hot spot regions in exons 7 and 8. Confirming these results, reported mutations of p53 in human culture cells, MiaPaCa2, TE-6, RPMI8226, DLD1, and PC-3 are detected at 5% relative abundance in the sample DNA.