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

J. Voisey, G. J. Hafner, C. P. Morris, A. Van Daal, P. M. Giffard

Research output: Contribution to journalArticle

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 derived from 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.

Original languageEnglish
Pages (from-to)1122-1129
Number of pages8
JournalBioTechniques
Volume31
Issue number5
DOIs
Publication statusPublished - 26 Nov 2001
Externally publishedYes

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