<Li> Circular template ligation, which can be conducted via template mediated enzymatic ligation (e.g., T4 DNA ligase) or template - free ligation using special DNA ligases (i.e., CircLigase). </Li> <Li> Primer - induced single - strand DNA elongation . Multiple primers can be employed to hybridize with the same circle . As a result, multiple amplification events can be initiated, producing multiple RCA products ("Multiprimed RCA"). A linear RCA product can be converted into multiple circles using restriction enzyme digestion followed by template mediated enzymatic ligation . </Li> <Li> Amplification product detection and visualization, which is most commonly conducted through fluorescent detection, with fluorophore - conjugated dNTP, fluorophore - tethered complementary or fluorescently - labeled molecular beacons . In addition to the fluorescent approaches, gel electrophoresis is also widely used for the detection of RCA product . </Li> <P> RCA can amplify a single molecular binding event over a thousandfold, making it particularly useful for detecting targets with ultra-low abundance . RCA reactions can be performed in not only free solution environments, but also on a solid surface like glass, micro - or nano - bead, microwell plates, microfluidic devices or even paper strips . This feature makes it a very powerful tool for amplifying signals in solid - phase immunoassay s (e.g., ELISA). In this way, RCA is becoming a highly versatile signal amplification tool with wide - ranging applications in genomics, proteomics, diagnosis and biosensing . </P>

Rolling circle model of dna replication in bacteria