IMPROVED DETECTION OF P53 POINT MUTATIONS BY DIDEOXYFINGERPRINTING (DDF)

Two screening techniques for identifying point mutations (single-stran d 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, Te n mutations were identified by sequencing; of these, 10/10 were detect ed by ddF, while SSCP detected 6/10 true mutations and falsely identif ied two presumed mutations not confirmed by sequencing. We examined th e impact of parameters which influence DNA conformation (gel temperatu re, 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 nucl eotides), We conclude that the increased sensitivity of ddF, together with its greater ease of application due to the lack of need for optim ization, provides significant advantages over SSCP in screening DNA se quences for the presence of point mutations, Our results also suggest that the incidence of p53 mutations may be underestimated in studies o f human cancers which utilize SSCP as the method of mutational screeni ng.