Novel polymerase chain reaction approach for full-coding p53 mutation detection in microdissected archival tumors

Evidence suggests that up to 25% of p53 mutations are outside of exons 5-8 and that insertions, deletions, and polymorphic sites in the p53 gene may p lay a significant role in the process of carcinogenesis. A novel polymerase chain reaction (PCR) approach for the analysis of the entire p53 coding an d splice site regions from microdissected, formalin-fixed, paraffin-embedde d tumor tissues has been developed which allows multiple genetic analyses t o be performed from one primary amplification reaction. The method was init ially evaluated using well-characterized cell lines. In addition to confirm ing the published p53 mutations for HT29, Molt 4, A431, and HN5, a 16 base pair (bp) duplication within intron 3 was detected in both the A431 and HT2 9 cell lines. Analysis of archival samples of ovarian cancer identified the same 16-bp duplication and coding region variations. In all samples, using GenBank submission U94788 as a reference, a C-insertion was detected at nu cleotide positions 11818 and 11874 within intron 2. At nucleotide position 14168. within intron 7, a T-to-G base change was found. This novel PCR appr oach has the potential to reduce the amount of clinical material required b y up to 95%, thus facilitating retrospective studies on archival tumor coll ections. Furthermore, a wider analysis of the p53 gene, including splice si tes and intronic regions, may yield additional information regarding cancer predisposition, response to therapy, and progression.