Mutation identification DNA analysis system (MIDAS) for detection of known mutations.
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 32p- 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 BRCA 1 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.
Bazar
L S
LS
Georgetown University, Department of Biochemistry, and Molecular Biology, Washington DC, USA.
Collier
G B
GB
Vanek
P G
PG
Siles
B A
BA
Kow
Y W
YW
Doetsch
P W
PW
Cunningham
R P
RP
Chirikjian
J G
JG
D019313:BRCA1 Protein / Q000235:genetics*; D020137:Base Pair Mismatch; D004273:DNA, Neoplasm / Q000032:analysis*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D019075:Electrophoresis, Capillary / Q000379:methods; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine; D006801:Humans; D007834:Lasers; D013941:Thymine
D019313:BRCA1 Protein; D004273:DNA, Neoplasm; D006147:Guanine; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D013941:Thymine