J. Voisey et al., Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (large fragment), BIOTECHNIQU, 31(5), 2001, pp. 1122
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 f
rom rolling circle replication of a circular template, the reaction is term
ed 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 a
n ELISA-like protocol. The circular template is derived by annealing and li
gation of a padlock on target DNA. It was found that there was good correla
tion between the synthesis of amplified material and signal. The specificit
y of the reaction with respect to single-nucleotide polymorphisms was inves
tigated, and it was found that Bst DNA polymerase is prone to extension fro
m primers with mismatched 3' ends. Reliable single nucleotide specificity w
as only obtained when pre-synthesized amplified material was interrogated b
y competitive primer extension.