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Mutation identification DNA analysis system (MIDAS) for detection of known mutations
Author(s) -
Bazar Leonard S.,
Collier G. Bruce,
Vanek Philip G.,
Siles Barbara A.,
Kow Yoke W.,
Doetsch Paul W.,
Cunningham Richard P.,
Chirikjian Jack G.
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:6<1141::aid-elps1141>3.0.co;2-7
Subject(s) - dna glycosylase , microbiology and biotechnology , dna , oligonucleotide , ap site , endonuclease , point mutation , biology , hybridization probe , chemistry , biochemistry , dna repair , mutation , gene
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 detection 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 endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32 P‐ or FITC‐labeled oligonucleotides as mutation probes. Generally between 20—50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full‐length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser‐induced fluorescence‐capillary electrophoresis (LIF‐CE) of fluorophore‐labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3′‐endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG‐MRE). MIDAS differentiated between a clinical sample BRCA1 wild‐type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF‐CE should make detection of known point mutations, deletions, and insertions a rapid and cost‐effective technique well suited for automation.