Cloning of Complete Geminivirus Genomes by Rolling Circle Amplification (RCA).

Rolling circle amplification (RCA) revolutionized geminivirology before high-throughput sequencing. Using the DNA polymerase of phage phi29 and random primers, it is a simple isothermal reaction which will amplify circular DNA without any prior knowledge of its sequence. It is thus ideal to clone complete geminivirus genomes. The procedure outlined here has been used in the author's laboratory for more than 15 years and has allowed the cloning of thousands of full-length genomes. It consists of three parts: first, the RCA reaction itself; second, the analysis of RCA products with restriction enzymes, to identify an enzyme that will cleave the genome at a single site; and third, the ligation of the linearized genome to a plasmid vector, followed by E. coli transformation. The whole procedure will normally take 3 days.