With the inception of PCR in the 1980s, the amplification of nucleic acid sequences has been a transformative tool in biological and biomedical research. In the decades since PCR’s introduction, other non-PCR amplification methods have been developed, opening the door for new applications for what may be impractical or unfeasible with PCR. Rolling circle amplification (RCA) is a non-PCR amplification technique that has been adopted in molecular diagnostics, biodetection, nanotechnology and other emerging areas. RCA differs from PCR in many ways. RCA is much more capable of detecting and amplifying low-abundance sequences. The technique utilizes unique DNA polymerases (most commonly Phi29) or T7 RNA polymerase to generate long single-stranded DNA or RNA products, respectively, by continuously amplifying a circular DNA template (generally ~15–200 nucleotides in length). The polymerases used for RCA are active at constant room temperature (37˚C), making thermal cycler instruments unnecessary. The resulting RCA product is composed of tens to hundreds of tandem repeats, complimentary to the circular template. RCA products can be detected by labeling or probing. RCA is an exceptionally sensitive technique, with the ability to detect specific single nucleotide variations in a DNA or RNA sequence. Because only precise target sequences are amplified, RCA is a powerful tool in SNP analysis and molecular diagnostics of genetic disease. RCA can also be used to detect non-NA biomolecules, such as proteins and analytes, by coupling with targeting molecules, such as antibodies. RCA assays are not limited to being performed in solution, as they can also be performed on solid-phase surfaces, such as glass, plates or beads if the molecule of interest is first captured on these scaffolds. The ability to create tandem repeating sequences has opened applications for RCA in nanobiotechnology and materials science. Synthesized sequences form the basis for constructing micro- and nano-structures with potential therapeutic and drug delivery uses. While RCA is suitable for some general amplification applications, such as whole genome amplification, it is mainly used in more niche applications and not likely to replace PCR for most amplification and sequencing purposes. Instead, it is a complementary tool with emerging applications in biodetection and bioengineering. RCA-based kits are produced by numerous vendors. Basic kits include a polymerase enzyme and buffer, while more inclusive kits include a ligase enzyme for creating circular DNA templates. Kits are also available for specific applications, such as whole genome amplification and single-cell amplification. Kit costs vary greatly depending on vendor, type of polymerase used and intended application. Basic kits can be found for as little as $60 for 100 reactions, while specific-purpose kits range from $300 to $550 for 100 reactions.
Rolling Circle Amplification at a Glance:
- GE Healthcare Life Sciences
- $60–$550 for 100 reactions