In its most fundamental format, a linear RCA (LRCA) requires a circular single-stranded oligonucleotide (DNA or RNA) as template , another single-stranded DNA or RNA primer that may also be the detection target , a strand displacement polymerase (e.g., 29 for ambient temperature and BST or vent(exo-) for 55 -65C (24-26)), dNTPs, and appropriate buffer. The circular ssDNA template can be either pre-synthesized (i.e., added to the reaction in the already circularized state), or prepared by in situ ligation prior to amplification. For assays using pre-synthesized circular DNA (cDNA), the traditional and most common method of preparing cDNA has been through splint-padlock ligation . In this method, an oligonucleotide "splint" (could be a DNA or RNA) anneals to another 5-phosphorylated oligonucleotide "padlock" to bring 5and 3-terminus of the latter to immediate proximity. A DNA or RNA ligase (depending on the splint) then joins the termini, creating a cDNA. Alternatively, a 5phosphorylated oligonucleotide can also be converted to a cDNA in a sequence selective manner using a CircLigase enzyme without using a splint . An alternative method of preparing cDNA involves self-annealing, where the 5-and 3-termini of a 5-phosphorylated oligonucleotide are brought in proximity by intramolecular annealing. The self-annealed ligation is then completed by a DNA ligase joining the termini. RCA assays involving selfannealed ligation have seen applications in the detection of leukemia biomarker BCR/ABLp210 fusion , and DNA methyltransferase activity . A third method of preparing cDNA involves cohesive end ligation, where the 5-and 3-termini are brought in proximity for ligation using complementary overhangs called "cohesive ends" or "sticky-ends" . Such cohesive ends can be generated by enzymatic digestion or deliberate sequence design, followed by ligation to enable circularization . RCA assays involving cohesive end ligated cDNAs have been applied for the detection of thrombin and let-7 family miRNA . Circular dumbbellshaped DNA can also be prepared by blunt-ended ligation of two hairpins, although this method produces symmetrical cDNA . The cDNA prepared by any of these methods can be purified from unligated oligonucleotide precursors by either exonuclease digestion or by denaturing polyacrylamide gel electrophoresis (PAGE) .