A DNA probe assay using strand displacement amplification (SDA) and filtration to separate reacted and unreacted detector probes

Strand displacement amplification (SDA) is an isothermal, in vitromethod of amplifying a target DNA sequence. We performed SDA in the presence of a 5- 32P-oligodeoxynucleotide detector probe that contains a target binding sequence at its 3-end and a recognition site for the restriction enzyme HincII at its 5-end which is not homologous to the target sequence. The single-stranded probe hybridizes to the rising concentration of amplified product during SDA and is converted to a fully double-stranded form that is cleaved by HincII, releasing a 32P-labelled 5-mer fragment. Uncleaved probe (42-mer) and cleaved probe (5-mer) were separated by either gel electrophoresis or size exclusion filtration using a commercially available microcentrifuge device. The combined SDA/filtration protocol is simple and provides detection of as few as 10 molecules of target DNA. We applied the technique to detection of M. tuberculosisDNA.