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

Abstract：

: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.

journal_name

Mol Cell Probes

journal_title

Molecular and cellular probes

authors

Walker GT,Nadeau JG,Linn CP

doi

10.1006/mcpr.1995.0062

subject

Has Abstract

pub_date

1995-12-01 00:00:00

pages

399-403

issue

6

eissn

0890-8508

issn

1096-1194

pii

S089085088570062X

journal_volume

9

pub_type

杂志文章

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