STRAND DISPLACEMENT AMPLIFICATION (SDA) AND TRANSIENT STATE FLUORESCENCE POLARIZATION DETECTION OF MYCOBACTERIUM-TUBERCULOSIS DNA

Strand displacement amplification (SDA) is an isothermal, in vitro met hod of amplifying a DNA sequence for diagnostic purposes. We have comb ined SDA with fluorescence polarization detection in a closed, homogen eous format. A fluorescently labeled oligodeoxynucleotide detect-or pr obe hybridizes to the amplification product that increases in concentr ation during SDA. The single- to double-stranded conversion of the pro be is accompanied by an increase in fluorescence polarization values, which can be measured in real-time without physical manipulation of th e sample, The probe was labeled with the near-infrared dye La Jolla Bl ue, and fluorescence polarization was measured on a transient-state fl uorometer, We have applied this homogeneous SDA/detection system to a target DNA sequence specific for Mycobacterium tuberculosis DNA.