Highly multiplex PCR assays by coupling the 5′-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters

Significance We describe a highly multiplex PCR approach that can identify 10-fold more targets in current real-time PCR assays without additional enzymes or separate reactions. This single-step, single-tube, homogeneous detection approach, termed MeltArray, is achieved by coupling the 5′-flap endonuclease activity of the Taq DNA polymerase and multiple annealing sites of the molecular beacon reporters. The 5′-flap endonuclease cleaves a probe specifically into a “mediator” primer, and one molecular beacon reporter allows for the extension of multiple “mediator” primers to produce a series of fluorescent hybrids with different melting temperatures unique to each target. The overall number of targets detectable per reaction is equal to the number of the reporters multiplied by the number of mediator primers per reporter.