RCA reactions have also been analyzed using intercalating dyes ( 19). Both methods require laborious handling of gels or microscope slides, and the precision of quantification is limited. The course of RCA reactions has been studied using electrophoresis ( 5, 7– 9), or by hybridizing labeled oligonucleotides to RCA products attached to a glass slide ( 5, 6, 17, 18). The combination of padlock probe circularization and amplification through RCA has proven useful for genetic analysis ( 5, 16). Padlock probes have been used to genotype samples of genomic DNA or RNA in solution ( 5, 11– 13), or for in situ genotyping of metaphase chromosomes and interphase nuclei ( 12, 14, 15). linear DNA probes that become circularized upon recognition of a specific nucleic acid sequence ( 10), since only reacted probes are amplified. RCA is particularly useful for signal amplification of padlock probes, i.e. The method is based on the finding that also small DNA circles can be copied by a rolling‐circle replication mechanism ( 5, 7– 9), known from the replication of phage genomes. Rolling‐circle amplification (RCA) has been applied for sensitive detection of nucleic acid sequences and of proteins ( 5, 6). This has been utilized to study the activity of single‐strand nucleases ( 3) and single‐strand binding proteins ( 4). The stem–loop structure can also be disrupted due to degradation or binding by proteins. Upon hybridization to a target nucleic acid molecule, the fluorophore is extended away from the quencher and can emit fluorescence. Molecular beacons are equipped with a fluorophore and a fluorescence quencher at either end, so that the fluorophore is brought close to the quencher when the free probes adopt a stem–loop structure. Hairpin‐shaped hybridization probes, so‐called molecular beacons, have been used to detect, distinguish or quantify amplification products of DNA or RNA target sequences ( 2). Most commonly the assays are based on fluorescence resonance energy transfer (FRET), or fluorescence by compounds that intercalate in nucleic acids. Real‐time measurements of the polymerase chain reaction (PCR) have enabled convenient quantification of target sequences over a wide concentration range by the use of homogenous detection assays for the amplification products ( 1). Received JanuRevised ApAccepted INTRODUCTION
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