SIGNAL AMPLIFICATION OF PADLOCK PROBES BY ROLLING CIRCLE REPLICATION

Circularizing oligonucleotide probes (padlock probes) have the potenti al to detect sets of gene sequences with high specificity and excellen t selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal ampl ification, We demonstrate here that in order for the reaction to proce ed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation, If t he target strand has a nearby free 3' end, then the probe-target hybri ds can be displaced by the polymerase used for replication. The displa ced probe can then slip off the target strand and a rolling circle amp lification is initiated, Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exo nucleolytic activity. We found the Phi 29 DNA polymerase to be superio r to the Klenow fragment in displacing the target DNA strand, and it m aintained the polymerization reaction for at least 12 h, yielding an e xtension product that represents several thousand-fold the length of t he padlock probe.