Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment)

Voisey, Joanne, Hafner, Gregory G., Morris, Charles P., van Daal, Angela M., & Giffard, Philip M. (2001) Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment). Biotechniques, 31(5), pp. 1126-1129.

Abstract

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.

Impact and interest:

2 citations in Scopus
1 citations in Web of Science®
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ID Code: 8475
Item Type: Journal Article
Refereed: Yes
Additional Information: For more information, please refer to the journal’s website (see hypertext link) or contact the author.
Author contact details: p.giffard@qut.edu.au
Additional URLs:
ISSN: 0736-6205
Divisions: Current > Research Centres > CRC for Diagnostics
Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2001 Eaton Publishing Company
Deposited On: 06 Jul 2007 00:00
Last Modified: 21 Apr 2010 16:53

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