AUTOMATED FLUORESCENT ANALYSIS PROCEDURE FOR ENZYMATIC MUTATION DETECTION

The Enzymatic Mutation Detection(TM) (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed b y electrophoretic detection. We report an automated procedure, using f luorescently labeled probe and quantitative analysis on the ABI Prism( TM) 377 DNA Sequencer, that improves on earlier methods (1, 2) by elim inating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses th e bacteriophage resolvase T-4 endonuclease VII, which cleaves the hete roduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation te chniques, the EMD method uses a single protocol to identify point muta tions, deletions, and insertions for all DNA fragments. Test DNA sampl es are assayed directly from PCR reactions, and fragments up to 4 kb i n size have been assayed successfully. A independent analysis on the p 53 tumor suppressor gene from clinical samples has shown 100% sensitiv ity and 94% specificity. Because the fluorescent EMD assay has been op timized for high signal-to-noise ratios, mutations can be identified i n mixed samples containing up to a 20-fold excess of normal DNA.