Detection of M. tuberculosisDNA using Thermophilic Strand Displacement Amplification

Abstract

Strand Displacement Amplification (SDA) is an isothermal, in vitromethod 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. colipolymerase I (exo -Klenow) and the restriction enzyme Hincll to achieve 10 8-fold amplification in 2 h at 37°C (Walker, G.T., 1993, PCR Methods and Applications 31–6). 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°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.