DETECTION OF MYCOBACTERIUM-TUBERCULOSIS DNA USING THERMOPHILIC STRANDDISPLACEMENT AMPLIFICATION

Strand Displacement Amplification (SDA) is an isothermal, in vitro met hod of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was devel oped which utilizes the exonuclease deficient Klenow fragment of E. co li polymerase I (exo(-)Klenow) and the restriction enzyme Hincll to ac hieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1 993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacill us stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient poly merase from Bacillus caldotenax (exo(-)Bca). SDA was used to amplify D NA from Mycobacterium tuberculosis. An amplification factor of 10(10)- fold was achieved after 15 min of SDA at 60 degrees C. The new thermop hilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification produ cts. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase. (C) 1996 Academic Press Limited