RAPID AND EFFICIENT SCREENING FOR P53 GENE-MUTATIONS BY DIDEOXY FINGERPRINTING

Dideoxy fingerprinting (ddF) is an efficient method for detecting sing le base and other sequence changes in PCR-amplified DNA segments. This screening method is a hybrid between single-strand conformation polym orphism analysis (SSCP) and Sanger dideoxy sequencing. It involves a S anger sequencing reaction with one dideoxynucleotide followed by non-d enaturing gel electrophoresis. We are using ddF to screen for mutation s in the p53 tumor suppressor gene in primary breast cancers. ddF dete cted more than 100 mutations in different regions of the gene, includi ng all types of single-base mutations and microdeletions/microinsertio ns of various sizes. Furthermore, ddF reliably detected heterozygous m utations, if the region of interest was screened in both directions. I n a blinded, prospective study, ddF detected all 25 mutations within e xons 4-10 and adjacent flanking intronic regions previously found by d irect sequencing. ddF was also useful in scoring two common polymorphi sms within the p53 gene. Guidelines for preventing false-positive and false-negative results are summarized.