Practical Applications of Rolling Circle Amplification of DNA Templates

Abstract

Rolling circle amplification was initially described as the mechanism by which a variety of viruses replicate their circular genomes (1–5). Since that time, a number of in vitro applications of the rolling circle mechanism have been described (6). The three basic in vitro forms of rolling circle amplification (RCA) can be distinguished by the number of primers used in a reaction (see Figure 1). In the linear form of RCA, a DNA circle is amplified by polymerase extension of a single complementary primer in an isothermal reaction. Up to 105 tandemly-repeated, concatemerized copies of the DNA circle are generated by each of these primers. The so-called exponential form of RCA uses a second DNA primer of identical sequence to the DNA circle. The third RCA format, multiply-primed RCA, employs a mixture of random primers and the same highly processive polymerase used in linear RCA. Each of these RCA formats is associated with unique sets of applications, which will be described in more detail below.