RCAT™ driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.

In the presence of two suitably designed primers, a geometric amplification occurs via DNA strand displacement and hyperbranching to generate 1012 or more copies of each circle in 1 hour.

In addition to grossly amplifying a signal, this method—called Exponential-RCAT (E-RCAT™)—is adequately sensitive to detect point mutations in genomic DNA.

If a single primer is used, RCAT generates in a few minutes a linear chain of thousands of tandemly linked DNA copies of a target covalently linked to that target. The linear RCAT approach permits both spatial resolution and accurate quantitation of a target.

DNA generated by either linear RCAT or E-RCAT can be labeled with fluorescent DNP-oligonucleotide tags (1) that hybridize to multiple sites in the product DNA sequences. The “decorated” DNA can then be condensed into a small point source by cross-linking with a multivalent anti-DNP IgM (RCAT-CACHET; Figure 2).

RCAT can be used with fluorophore combinations designed for multiparametric color coding, thereby markedly increasing the number of targets that can be analyzed simultaneously. RCAT can be used in solution, in situ, and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules.