1. The circular DNA template used in the RCA process can be synthesized enzymatically or chemically through the intramolecular ligation of phosphate and hydroxy end groups. In the enzymatic synthesis (see Ref. [15] for a typical protocol), the two reactive ends of a linear DNA precursor are joined by an enzyme (e.g., T4 DNA ligase). The enzymatic synthesis proceeds efficiently (>90 % yield) for relatively large DNA substrates, but may not be suitable for making small circular DNA (<30 nucleotides (nt), and particularly those <10 nt), presumably as a result of inadequate saturation of the enzyme binding sites and/or the strain induced upon the ring closure of short oligonucleotides (see Refs. [1b-d]). This shortcoming might be an obstacle to the use of the RCA method in some cases, as enzymatic synthesis is the most commonly used strategy for the creation of the circular DNA template in RCA-based diagnostic assays. However, the chemical (or nonenzymatic) cyclization of DNA oligonucleotides can also be used to produce circular DNA molecules in reasonably good yield (up to 85 %) for both small (<14 nt; see Refs. [1e,f]) and large circular templates (>15 nt; see Ref. [1b]). For example, chemical ligation was used to produce a circular DNA molecule with 13 nt: the smallest circular DNA molecule studied to date that can be used effectively in an RCA process (see Ref. [1g]).
2. Convergent DNA synthesis: a non-enzymatic dimerization approach to circular oligodeoxynucleotides
3. T4 RNA ligase: Substrate chain length requirements
4. Catalysis of DNA joining by bacteriophage T4 RNA ligase