Mutation identification DNA analysis system (MIDAS) for detection of knownmutations

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA de tection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/apurinic (AP) site is cleaved by AP e ndonucleases/lyases either associated with the apurinic (AP) site is cleave d by AP endonucleases/lyases either associated with the Biochemistry, and D NA glycosylase or externally added to the reaction mixture. MIDAS utilizes P-32- Or FITC-labeled oligonucleotides as mutation probes. Generally betwee n 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the pr obe at the point of mismatch. Once the probe is cleaved, the fragments beco me thermally unstable and fall off the target, thereby allowing another ful l-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separ ation followed by autoradiography, or by laser-induced fluorescence-capilla ry electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes us ing a novel CE matrix. In the present experiments, we employed the mesophil ic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a nov el thermostable T/G DNA gycosylase, TDG mismatch repair enzyme (TDG-MRE). M IDAS differentiated between a clinical sample BRCA1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (P CR). The combination of MIDAS with LIF-CE should make detection of known po int mutations, deletions, and insertions a rapid and cost-effective techniq ue well suited for automation.