Automated fluorescent analysis procedure for enzymatic mutation detection

Abstract

The Enzymatic Mutation Detection (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism 377 DNA Sequencer, that improves on earlier methods (1, 2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.