We now present a procedure that integrates highly specific and sensitive recognition of target nucleic acid sequences using padlock probes with efficient local signal amplification, thereby permitting detection of single-nucleotide variation of individual DNA molecules in situ. In the process, amplification products risk drifting away, however, potentially further reducing the efficiency of localized detection. Upon addition of a primer the probes can be replicated in situ by RCA, but only if they escape the topological block against replication by detaching from the target molecules. In an interesting approach, target sequences were enzymatically rendered single stranded having free 5′ ends near the binding sites for padlock probes 15. Regrettably, replication of circular probes that remain wound around their target strands seems to be inhibited, probably as a consequence of molecular crowding 16, although there is some controversy in the literature about this topological inhibition 17. If, instead, the reaction is set up so that the RCA template can form only in a target-specific circularization reaction in situ, nonspecific signals should be greatly diminished and promising results have been reported with this approach 14, 15. Although this results in clearly detectable signals from individual probes, the specificity of the reaction may be insufficient for robust detection of unique single-copy sequences in situ because of difficulties in distinguishing specifically bound from nonspecifically bound probes. More recently, linear probes hybridized in situ have been extended not by replicating the target sequence, but by using separately added small circular DNA strands as templates for a localized rolling-circle amplification (RCA) 12, 13. This process enables localized allele-specific detection of repeated sequences in the genome 8, 9, 10, 11, but this method, too, falls short of detecting single-copy genes from single primers, as would be needed for genotyping in situ. Linear oligonucleotide probes hybridized in situ have been used as primers for site-specific DNA synthesis, allowing labeled nucleotides to be incorporated in the so-called PRINS reaction 7. Unfortunately, detection signals from specifically circularized padlock probes have proven insufficient to reveal single-copy gene sequences owing to background noise from nonspecifically bound probes, although repeated centromeric sequences have been evaluated with sufficient precision to investigate the distribution of single-nucleotide variants in situ 5, 6. These probes have detection specificity similar to that obtained with PCR 1, 2, 3, but unlike in PCR, the reaction products can remain bound to their target sequences, even withstanding denaturing washes 4. Oligonucleotide probes with one target-complementary sequence at each end-padlock probes-encircle specific target molecules as a consequence of templated DNA ligation. Unlike simple hybridization probes, PCR provides adequate specificity to detect unique target sequences in the context of whole genomes, but the technique is poorly suited for precisely localized detection reactions. By analyzing biological processes at the ultimate level of single molecules, and with sufficient precision to distinguish even closely similar variants, it will be possible to study the inter- or subcellular context of specific DNA or RNA sequences as well as to analyze their location among extensive, arrayed sets of biological samples.
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