Skip to main content
Article
Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (large fragment)
BioTechniques (2001)
  • J Voisey
  • G Hafner
  • C Morris
  • Angela Van Daal, Queensland University of Technology
  • P Giffard
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.
Disciplines
Publication Date
November, 2001
Publisher Statement

Interim status: Citation only.

Voisey J., Hafner, G., Morris, C., van Daal, A. and Giffard, P. (2001) Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (large fragment). BioTechniques, Vol. 31, Iss. 5, pp. 1122-1129.

View BioTechniques homepage.

Copyright ©Eaton Publishing Company, 2001.

Citation Information
J Voisey, G Hafner, C Morris, Angela Van Daal, et al.. "Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (large fragment)" BioTechniques Vol. 31 Iss. 5 (2001)
Available at: http://works.bepress.com/angela_v_daal/13/