Figure 2

ERCA using Q-PNA, P1 and P2 Exponential amplification of circularized OCP was performed using primers P1 and P2. P1 is a composite of a Q-PNA annealed to a fluorescent DNA primer specific for the circle sequence directed against one allele. Q-PNA anneals to the fluorescent DNA primer such that the c-terminal quencher on the PNA quenches the 5' fluorescent moiety (either FAM or Cy3) on the DNA primer. P2 is a DNA primer with the same strand orientation as the circle, complementary to the product of the primary rolling circle reaction. Amplification of the circle specific for only one of the two alleles is illustrated. ERCA begins when P1 anneals to the circle (1), and polymerase copies the circle sequence, eventually copying the entire circle. The polymerase begins displacing the previously synthesized product, opening up single stranded P2 binding sites (2). Each turn of displacement synthesis around the circle exposes another P2 binding site, and the resulting synthesis from the annealed P2 primers results in the displacement of downstream primers and products, including the PNA (3). Displacement of the PNA separates the fluorescent label on the DNA from the quencher on the PNA, producing fluorescent signal specific for the P1/circle pair (4). Displacement of the downstream primers and products also opens up additional P1 binding sites, and the process continues in an exponential cascade.