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

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.