More keys to padlock probes: mechanisms for high-throughput nucleic acid analysis.

With the impending availability of total information about nucleic acid sequences in humans and other organisms, tools to investigate these sequences on a large scale assume increasing importance. Methods currently in use, however, cannot offer the required combination of high-throughput, sensitivity and specificity of detection. Padlock probes, circularizing oligonucleotides, may provide a means to detect, distinguish, quantitate and also locate very large numbers of DNA or RNA sequences. Recent developments in areas such as the biochemistry of ligation and characterization of ligases, methods to replicate circularized probes and the development of assays based on these principles augment the potential of padlock probes.

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent 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 amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.

A manifold support for molecular genetic reactions.

Large numbers of samples can be efficiently processed through sequential reaction steps using a 96-pronged support that projects into individual microtiter wells. The support was constructed by creating a porous surface on a disposable polystyrene manifold, with avidin coupled to this greatly expanded surface. The shape and high binding capacity of the device allow the parallel transfer of large sets of reaction intermediates between different binding or enzymatic processing steps. We have applied the support to increase the efficiency of preparative and analytical molecular genetic reactions. The support also reduce the risks of sample mix-up and contamination in applications such as PCR and DNA sequencing.