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

• Published in: Molecular and cellular probes Vol. 9; no. 6; pp. 399 - 403
• Main Authors: Walker, Terrance G., Nadeau, James G., Linn, Preston C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-12-1995
• Subjects: Base Sequence; Centrifugation; Deoxyribonucleases, Type II Site-Specific; DNA - genetics; DNA - metabolism; DNA Polymerase I; DNA Probes - analysis; DNA Probes - isolation & purification; DNA Probes - metabolism; DNA, Bacterial - analysis; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; DNA, Single-Stranded - genetics; Electrophoresis, Polyacrylamide Gel; M. tuberculosis; Microchemistry; Molecular Sequence Data; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Nucleic Acid Amplification Techniques; Nucleic Acid Denaturation; Phosphorus Radioisotopes - analysis; strand displacement amplification (SDA); Ultrafiltration; M. tuberculosis; strand displacement amplification (SDA)
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1006/mcpr.1995.0062

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.