Detection of M. tuberculosis DNA using thermophilic strand displacement amplification.
Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.
Spargo
C A
CA
Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709, USA.
Fraiser
M S
MS
Van Cleve
M
M
Wright
D J
DJ
Nycz
C M
CM
Spears
P A
PA
Walker
G T
GT
D001407:Bacillus / Q000201:enzymology; D004269:DNA, Bacterial / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D013696:Temperature; D014544:Uridine Triphosphate
D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D014544:Uridine Triphosphate