DNA diagnosis with mutation-specific artificial methylation sites: application to rapid screening of delta F508.
Many polymerase chain reaction (PCR)-based methods for diagnosis of minute mutations are suboptimal for automated screening because of their reliance on gel electrophoresis or probe hybridization. In the method reported here, PCR products containing artificial methylation sites are analyzed by measuring incorporation of radiolabeled methyl groups. Primers are designed to amplify the possible mutation-containing region such that the 3' end of one primer lies adjacent to the possible mutation. Sequence modification near that end creates either a mutation- or wild type (WT)-specific artificial methylation site in the PCR product. The product is briefly incubated with an appropriate DNA methylase and tritiated S-adenosylmethionine ([3H]SAM), separated from free SAM by column chromatography, and analyzed for incorporation of tritium. Applying this technique to the cystic fibrosis delta F508 deletion, we accurately diagnosed five homozygotes, five heterozygotes, and five normal individuals within 40 min of PCR completion. The method can be generalized to rapid, automated detection of a variety of point mutations and small deletions.
Petty
E M
EM
Department of Genetica, Yale University School of Medicine, New Haven, CT 06510.
Gold
E
E
Bale
A E
AE
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D004247:DNA / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific; D017353:Gene Deletion; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004247:DNA; C032824:ATCGAT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific