Mutation detection and single-molecule counting using isothermal rolling-circle amplification

Nat Genet. 1998 Jul;19(3):225-32. doi: 10.1038/898.


Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Base Sequence
  • Cell Line
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • DNA Mutational Analysis / methods*
  • DNA Probes
  • DNA, Circular
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques*
  • Point Mutation


  • CFTR protein, human
  • DNA Probes
  • DNA, Circular
  • Cystic Fibrosis Transmembrane Conductance Regulator