MUTATION DETECTION AND SINGLE-MOLECULE COUNTING USING ISOTHERMAL ROLLING-CIRCLE AMPLIFICATION

Rolling-circle amplification (RCA) driven by DNA polymerase can replic ate circularized oligonucleotide probes with either linear or geometri c kinetics under isothermal conditions. In the presence of two primers , one hybridizing to the + strand, and the other, to the - strand of D NA, 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 g enerates hundreds of tandemly linked copies of a covalently closed cir cle in a few minutes. If matrix-associated, the DNA product remains bo und at the site of synthesis, where it may be tagged, condensed and im aged as a point light source. Linear oligonucleotide probes bound cova lently on a glass surface can generate RCA signals, the colour of whic h indicates the allel status of the target, depending on the outcome o f specific, target-directed ligation events. As RCA permits millions o f individual probe molecules to be counted and sorted using colour cod es, it is particularly amenable for the analysis of rare somatic mutat ions. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.