Methods are needed to identify large numbers of nucleic acid sequence variations and to clarify their biological significance. We are developing a method for identification and quantitation of DNA and RNA sequences using padlock probes. The two ends of these linear DNA probes can hybridize next to each other on a target strand. Only when the ends are correctly matched to the target, however, can they be joined by ligation, converting the probes to circular molecules. Specific nucleic acid sequences can be detected by scoring circularized probes. We have shown that padlock probes are sufficiently specific to distinguish single-nucleotide variation in total genomic DNA. If only circularized probes can be amplified, it may be possible to perform highly multiplex analysis, because only intramolecular reactions are detected. Such a method would be an alternative to genotyping methods relying on multiplex PCR. We are applying a rolling-circle replication mechanism to amplify circularized probes and achieve localized signal amplification for parallel analysis of sets of target sequences. The method employs arrayed primers that bind specific padlock probes and initiate rolling-circle replication. A localized, linear concatemer is generated that is detected by fluorescence microscopy in a microarray format. Recently we have shown that padlock probes can also be ligated on RNA template with good sequence discrimination. Therefore, this method may also be suitable for analysis of RNA expression. The ability of padlock probes to detect RNA sequence variation may facilitate the analysis of closely related genes and alternatively spliced transcripts, which have been difficult to study with cDNA-based expression arrays. The inherent signal amplification will aid in quantitation of low-abundance RNA, and may expand the use of expression arrays by permitting the study of small cell populations such as developing tissues and solid tumours.
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