Improved detection of p53 point mutations by dideoxyfingerprinting (ddF).
Two screening techniques for identifying point mutations (single-strand conformational polymorphism (SSCP) and dideoxyfingerprinting (ddF)) were compared to sequencing to determine their efficiency in detecting mutations in exons 5-8 of the p53 tumor suppressor gene. Twelve human glioblastoma cell lines were studied by each of the three methods. Ten mutations were identified by sequencing; of these, 10/10 were detected by ddF, while SSCP detected 6/10 true mutations and falsely identified two presumed mutations not confirmed by sequencing. We examined the impact of parameters which influence DNA conformation (gel temperature, gel composition, and PCR product size) on the ability of SSCP and ddF to detect mutations. The sensitivity of SSCP varied with both gel temperature and the size of the PCR product; in contrast, ddF was not influenced by either gel temperature or product length (up to 460 nucleotides). We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optimization, provides significant advantages over SSCP in screening DNA sequences for the presence of point mutations. Our results also suggest that the incidence of p53 mutations may be underestimated in studies of human cancers which utilize SSCP as the method of mutational screening.
Martincic
D
D
Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6310, USA.
Whitlock
J A
JA
D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D015224:Dideoxynucleosides / Q000737:chemistry*; D004586:Electrophoresis / Q000379:methods; D016158:Genes, p53; D005909:Glioblastoma / Q000235:genetics / Q000473:pathology; D006801:Humans; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000235:genetics*
D015224:Dideoxynucleosides; D016159:Tumor Suppressor Protein p53; D004247:DNA