The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.