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 vitro method 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. tuberculosis DNA.
Walker
G T
GT
Becton Dickinson Research Center, Research Triangle Park, NC 27709-2016, USA.
Nadeau
J G
JG
Linn
C P
CP
D001483:Base Sequence; D002498:Centrifugation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I; D015342:DNA Probes / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D008832:Microchemistry; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques; D009691:Nucleic Acid Denaturation; D010761:Phosphorus Radioisotopes / Q000032:analysis; D014462:Ultrafiltration
D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D010761:Phosphorus Radioisotopes; D004247:DNA; D004256:DNA Polymerase I; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases