Written and maintained by CASRAI Editorial Board
Last updated
Rolling circle amplification (RCA) is an isothermal enzymatic method that copies a small circular nucleic acid template into one long single-stranded molecule, using a strand-displacing polymerase such as phi29 at 25-37 °C with no temperature cycling. It is used in diagnostics and in-situ detection, and it differs from PCR in both product and equipment.
Short answer
RCA turns a circular DNA template into a long single-stranded product made of tandem repeats of that circle’s sequence. A strand-displacing polymerase keeps running around the circle, so one binding event can be amplified more than a thousand-fold, and the reaction runs at a constant temperature, so no thermal cycler is needed.
Definition and scope
A review of RCA in integrated microsystems describes it as allowing “the unidirectional amplification of a circular nucleic acid template into a long single stranded molecule” (Soares, Madaboosi and Nilsson, Accounts of Chemical Research, 2021). A review of RCA-based biomedical sensing describes an isothermal enzymatic process that uses DNA or RNA polymerases and produces a long single-stranded DNA with hundreds to thousands of repeat units (Gu et al., Pharmaceuticals, 2018). The reaction relies on a strand-displacing polymerase, which is why no thermal cycling is involved.
The product is a concatemer: a single long strand carrying the target sequence in tandem repeats, not a pool of separate copies of the circle. That structure is what makes RCA useful for detection, because each product concentrates many binding sites for labelled probes in one place.
How RCA is used in practice
The published reviews describe the same overall arrangement, and the order of the stages matters more than any single reagent.
1. A circle is made, often by a padlock probe
RCA needs a circular template. In padlock-probe designs, a linear probe whose two ends hybridize to the target is cyclized by a ligase once it is bound; ligation “enables target-specific probe circularization” (Soares et al., 2021). Because the circle forms only when the probe matches the target, the padlock mechanism gives single-base specificity in the same review’s account.
2. A polymerase runs around the circle
Phi29 DNA polymerase is the enzyme named in both the 2018 and 2021 reviews; the 2021 review says it has the best processivity and strand-displacement activity and can amplify a 100 bp circular probe into a DNA concatemer (about 1,000 copies in 1 hour, per that review). The 2021 review lists conditions of 25-37 °C, isothermal.
3. The product is read out
For detection, complementary labelled oligonucleotides are added and each product binds many of them. The 2018 review lists readouts including surface plasmon resonance, electrochemical, fluorescence and colorimetric methods (the last using a G-quadruplex/hemin DNAzyme). RCA also appears inside other assays: in the proximity ligation assay, it is the amplification step.
Related concepts
- Padlock probe RCA: the probe is circularized on the target, then amplified (Soares et al., 2021; Gu et al., 2018).
- Hyperbranched RCA (HRCA): the 2018 review notes that Bst DNA polymerase is used in this variant. A 2026 protocol for sequencing extrachromosomal circular DNA also describes its output as hyper-branched structures.
- Two rounds of RCA: the 2021 review states that two rounds can give a million-fold amplification of the initial template, assuming about 1,000 circle copies per round; this is a theoretical estimate, not a measured result.
- Multiplexing: by sequential probing and stripping, the 2021 review reports extension to more than 200 simultaneous target sequences.
Where it matters for labs and supply decisions
RCA is a low-equipment method: because the reaction is isothermal and needs no temperature cycling (Gu et al., 2018), the reagent list, rather than a thermal cycler, defines the workflow. One published protocol, for RCA-based nanopore sequencing of extrachromosomal circular DNA from cell and plasma samples (STAR Protocols, 2026), shows what a working reagent list looks like. Its key resources table lists the following categories, which makes a useful checklist when scoping any RCA workflow:
| Item category | Listed in the protocol’s key resources |
|---|---|
| Polymerase | A phi29-type DNA polymerase (Phi29 MAX DNA Polymerase in that protocol) |
| Primers | Exo-random primers |
| Nucleotides | dNTPs, supplied as a 10 mM mix |
| Additives | Recombinant albumin and inorganic pyrophosphatase |
| Clean-up | SPRI (magnetic) beads |
| Quantification | A fluorometric dsDNA high-sensitivity assay kit |
The same protocol runs the amplification at 30 °C and uses a 65 °C heat inactivation, which sits inside the isothermal range given in the 2021 review. A padlock-probe design instead needs a ligase and a target-specific probe, which this protocol does not use, so the reagent list depends on the format.
For related bench work, see PCR protocol basics and the qPCR and RT-qPCR guide for thermocycled quantification, and digital PCR vs qPCR for how absolute and relative quantification differ.
Common misunderstandings
RCA is not PCR
PCR cycles through temperatures; RCA is isothermal and needs no temperature-cycling device (Gu et al., 2018; Soares et al., 2021). The products also differ: RCA yields one long single strand of tandem repeats, not discrete double-stranded amplicons.
The product is not separate copies of the circle
The concatemer is one continuous strand, which is why labelled probes can concentrate many binding sites on a single product.
Isothermal does not mean enzyme-free or ligation-free
RCA still depends on a polymerase, and padlock-probe formats still depend on a ligase to close the circle. The 2021 review also states that RCA compatibility with minimally processed samples is still largely unexplored, and that direct RNA detection and sample-matrix effects still need work. The 2018 review notes that the RCA-electrochemistry designs it covers are complex compared with other designs.
FAQ
What does rolling circle amplification produce?
A long single-stranded DNA with tandem repeats of the target sequence, with hundreds to thousands of repeat units according to Gu et al. (2018).
Which enzyme is used for RCA?
Phi29 DNA polymerase is the example named in the 2018 and 2021 reviews, valued for processivity and strand displacement. Bst DNA polymerase is cited for hyperbranched RCA.
What temperature does RCA run at?
The 2021 review lists 25-37 °C, isothermal. The 2026 protocol above runs at 30 °C.
How much amplification does RCA give?
A single binding event can be amplified over a thousand-fold (Gu et al., 2018), and two rounds can reach about a million-fold (Soares et al., 2021).
Sources
Links checked 2026-10-29.
- Soares RRG, Madaboosi N, Nilsson M. Rolling Circle Amplification in Integrated Microsystems: An Uncut Gem toward Massively Multiplexed Pathogen Diagnostics and Genotyping. Acc Chem Res 2021;54(21):3979-3990. https://pmc.ncbi.nlm.nih.gov/articles/PMC8567418/
- Gu L, Yan W, Liu L, Wang S, Zhang X, Lyu M. Research Progress on Rolling Circle Amplification (RCA)-Based Biomedical Sensing. Pharmaceuticals (Basel) 2018;11(2):35. https://pmc.ncbi.nlm.nih.gov/articles/PMC6027247/
- Protocol for rolling circle amplification-based nanopore sequencing of eccDNA from cell and plasma samples. STAR Protocols 2026. https://pmc.ncbi.nlm.nih.gov/articles/PMC13659461/
