Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Rolling circle amplification as isothermal gene amplification in molecular diagnostics


Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations.


  1. 1.

    Fire, A. & Xu, S.Q. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645 (1995).

  2. 2.

    Liu, D. et al. Rolling Circle DNA Synthesis: Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases. J. Am. Chem. Soc. 118, 1587–1594 (1996).

  3. 3.

    Blanco, L. & Salas, M. Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication. Proc. Natl. Acad. Sci. USA 81, 5325–5329 (1984).

  4. 4.

    Blanco, L. et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940 (1989).

  5. 5.

    Beyer, S., Nickels, P. & Simmel, F.C. Periodic DNA nanotemplates synthesized by rolling circle amplification. Nano Lett. 5, 719–722 (2005).

  6. 6.

    Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085–2088 (1994).

  7. 7.

    Nilsson, M. Lock and roll: single-molecule genotyping in situ using padlock probes and rolling-circle amplification. Histochem. Cell Biol. 126, 159–164 (2006).

  8. 8.

    Lizardi, P.M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

  9. 9.

    Cheng, Y. et al. Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew. Chem. Int. Ed. Engl. 48, 3268–3272 (2009).

  10. 10.

    Schweitzer, B. & Kingsmore, S. Combining nucleic acid amplification and detection. Curr. Opin. Biotechnol. 12, 21–27 (2001).

  11. 11.

    Cheng, W. et al. A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification. Biosens. Bioelectron. 62, 274–279 (2014).

  12. 12.

    Faruqi, A.F. et al. High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification. BMC Genomics 2, 4 (2001).

  13. 13.

    Heo, H.Y. et al. A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification. Biosens. Bioelectron. 78, 140–146 (2016).

  14. 14.

    Li, J. et al. Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Anal. Chem. 82, 2811–2816 (2010).

  15. 15.

    Li, X. et al. Genotyping of multiple single nucleotide polymorphisms with hyperbranched rolling circle amplification and microarray. Clin. Chim. Acta 399, 40–44 (2009).

  16. 16.

    Pickering, J. et al. Integration of DNA ligation and rolling circle amplification for the homogeneous, endpoint detection of single nucleotide polymorphisms. Nucleic Acids Res. 30, e60 (2002).

  17. 17.

    Zhang, S., Wu, Z., Shen, G. & Yu, R. A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue. Biosens. Bioelectron. 24, 3201–3207 (2009).

  18. 18.

    Jonstrup, S.P., Koch, J. & Kjems, J. A microRNA detection system based on padlock probes and rolling circle amplification. RNA 12, 1747–1752 (2006).

  19. 19.

    Zhou, Y. et al. A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection. Nucleic Acids Res. 38, e156 (2010).

  20. 20.

    Mashimo, Y., Mie, M., Suzuki, S. & Kobatake, E. Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay. Anal. Bioanal. Chem. 401, 221–227 (2011).

  21. 21.

    Sun, Y., Gregory, K.J., Chen, N.G. & Golovlev, V. Rapid and direct microRNA quantification by an enzymatic luminescence assay. Anal. Biochem. 429, 11–17 (2012).

  22. 22.

    Li, Y., Liang, L. & Zhang, C.Y. Isothermally sensitive detection of serum circulating miRNAs for lung cancer diagnosis. Anal. Chem. 85, 11174–11179 (2013).

  23. 23.

    Liu, H. et al. High specific and ultrasensitive isothermal detection of microRNA by padlock probe-based exponential rolling circle amplification. Anal. Chem. 85, 7941–7947 (2013).

  24. 24.

    Zhang, L.R., Zhu, G. & Zhang, C.Y. Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification. Anal. Chem. 86, 6703–6709 (2014).

  25. 25.

    Zhuang, J., Lai, W., Chen, G. & Tang, D. A rolling circle amplification-based DNA machine for miRNA screening coupling catalytic hairpin assembly with DNAzyme formation. Chem. Commun. (Camb) 50, 2935–2938 (2014).

  26. 26.

    Miao, P. et al. Ultrasensitive detection of microRNA through rolling circle amplification on a DNA tetrahedron decorated electrode. Bioconjug. Chem. 26, 602–607 (2015).

  27. 27.

    Zhang, X. et al. Chemiluminescence detection of DNA/ microRNA based on cation-exchange of CuS nanoparticles and rolling circle amplification. Chem. Commun. (Camb) 51, 6952–6955 (2015).

  28. 28.

    Chen, Y. et al. A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs. Chem. Commun. (Camb) 51, 6980–6983 (2015).

  29. 29.

    Hong, C. et al. Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide. Anal. Chem. 88, 2999–3003 (2016).

  30. 30.

    Schopf, E. et al. Mycobacterium tuberculosis detection via rolling circle amplification. Anal. Methods 3, 267–273 (2010).

  31. 31.

    Fu, Z., Zhou, X. & Xing, D. Sensitive colorimetric detection of Listeria monocytogenes based on isothermal gene amplification and unmodified gold nanoparticles. Methods 64, 260–266 (2013).

  32. 32.

    Gomez, A., Miller, N.S. & Smolina, I. Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes. Anal. Chem. 86, 11992–11998 (2014).

  33. 33.

    Xiang, Y. et al. Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor. Biosens. Bioelectron. 66, 512–519 (2015).

  34. 34.

    Guo, Y. et al. Label-free and highly sensitive electrochemical detection of E. coli based on rolling circle amplifications coupled peroxidase-mimicking DNAzyme amplification. Biosens. Bioelectron. 75, 315–319 (2016).

  35. 35.

    Wang, B. et al. Rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification. J. Clin. Microbiol. 43, 2339–2344 (2005).

  36. 36.

    Brasino, M.D. & Cha, J.N. Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing. Analyst 140, 5138–5144 (2015).

  37. 37.

    Hamidi, S.V. & Ghourchian, H. Colorimetric monitoring of rolling circle amplification for detection of H5N 1 influenza virus using metal indicator. Biosens. Bioelectron. 72, 121–126 (2015).

  38. 38.

    Hamidi, S.V., Ghourchian, H. & Tavoosidana, G. Real-time detection of H5N 1 influenza virus through hyperbranched rolling circle amplification. Analyst 140, 1502–1509 (2015).

  39. 39.

    Rockett, R. et al. Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV. J. Virol. Methods 215-216, 17–21 (2015).

  40. 40.

    Esquela-Kerscher, A. & Slack, F.J. Oncomirs -micro RNAs with a role in cancer. Nat. Rev. Cancer 6, 259–269 (2006).

  41. 41.

    Ambros, V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113, 673–676 (2003).

  42. 42.

    Yanaihara, N. et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9, 189–198 (2006).

  43. 43.

    Rabinowits, G. et al. Exosomal microRNA: a diagnostic marker for lung cancer. Clin. Lung Cancer 10, 42–46 (2009).

  44. 44.

    Lu, Y.F. et al. IFNL 3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance. Sci. Rep. 5, 16037 (2015).

  45. 45.

    Schroder, N.W., Schumann, R.R. Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious disease. Lancet Infect Dis 5, 156–164 (2005)

  46. 46.

    Roses, A.D. Pharmacogenetics and the practice of medicine. Nature 405, 857–865 (2000).

  47. 47.

    Pharoah, P.D.P., Dunning, A.M., Ponder, B.A.J. & Easton, D.F. Association studies for finding cancer-susceptibility genetic variants. Nat. Rev. Cancer 4, 850–860 (2004).

  48. 48.

    Freitag, C.M. The genetics of autistic disorders and its relevance: a review of the literature. Mol. Psychiatry 12, 2–22 (2007).

  49. 49.

    Zhernakova, A., Diemen, C.C.V. & Wijmenga, C. Detecting shared pathogenesis from the shared genetics of immune-related diseases. Nat. Rev. Genet. 10, 43–55 (2009).

Download references

Author information

Correspondence to Dong-Eun Kim.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Goo, N., Kim, D. Rolling circle amplification as isothermal gene amplification in molecular diagnostics. BioChip J 10, 262–271 (2016).

Download citation


  • Rolling circle amplification
  • Isothermal DNA amplification
  • Molecular diagnostics
  • Micro RNA
  • Single Nucleotide Polymorphisms