DNA BASE MISMATCH DETECTION USING FLOW CYTOMETRY

摘要

Flow cytometric DNA base mismatch detection. The use of immobilized mismatchbinding protein-coated microspheres to bind fluorescently labeled, mismatchcontaining DNA for detection by flow cytometry is described. The genomic DNA of interest is made fluorescent by one of a number of techniques. The resulting fluorescent DNA is melted and allowed to reanneal. If the genomic sample contains a polymorphic site, the reannealed DNA will contain base mismatches. Microspheres bearing immobilized mismatch-binding protein are added to the DNA samples and the mismatch-containing DNA allowed to bind to the mismatch-binding proteins. The sample is then analyzed by flow cytometry to detect the fluorescent DNA bound to the microspheres. The present flowcytometric method for mutation scanning has several important features: it is a single step, no wash assay, capable of rapid and sensitive analysis. It will allow amplified regions to be rapidly scanned for the presence of polymorphisms to prioritize samples for complete sequencing or to detect mutations in known regions of the genome.