Rolling Circle Amplification (RCA)

Rolling circle amplification (RCA) is a method of isothermal amplification of circular DNA molecules. RCA assay involves DNA amplification using a DNA ɸ polymerase with strand displacement activity to extend a single or multiple primers annealed to a circular DNA template. The strand displacement activity allows the newly synthesized DNA template to displace the previously generated DNA molecule releasing single-stranded DNA (ssDNA). This enzymatic process of primer extension combined with DNA strand displacement generates a long single-stranded DNA containing a repeated sequence complementary to the circular DNA template. The method is successfully used for the detection of many circular single- and double-stranded DNA viruses (begomovirus, badnavirus) infecting different crops. In this chapter, different steps involved in the RCA of a begomovirus virus genome are discussed.