title: Detection of M. tuberculosis DNA using thermophilic strand displacement amplification.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Spargo
authors.forename: M S
authors.initials: MS
authors.lastname: Fraiser
authors.forename: M
authors.initials: M
authors.lastname: Van Cleve
authors.forename: D J
authors.initials: DJ
authors.lastname: Wright
authors.forename: C M
authors.initials: CM
authors.lastname: Nycz
authors.forename: P A
authors.initials: PA
authors.lastname: Spears
authors.forename: G T
authors.initials: GT
authors.lastname: Walker
mesh_terms: 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
chemical_list: D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D014544:Uridine Triphosphate