Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene

The ability to sequence a large number of DNA samples rapidly and accuratel y for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequenci ng(TM) is a non-electrophoretic real-time DNA sequencing method that uses t he luciferase-luciferin light release as the detection signal for nucleotid e incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DN A samples from skin-cancer patients were used. Two forms of nucleotide disp ensation strategy were used, cyclic and programmed. Bi-directional pyrosequ encing was performed and the overlapping sequence data produced were assemb led to determine the sequence of the gene. Reliable sequencing data were ob tained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequenci ng for detection of p53 mutations and allele distribution was demonstrated. (C) 2000 Elsevier Science B.V. All rights reserved.