MNAzyme qPCR with superior multiplexing capacity

MNAzymes (nucleic acid enzymes formed from multiple partial enzymes) can be linked to PCR to provide a highly specific method for target detection and quantification. We investigated the feasibility of multiplexing MNAzyme quantitative PCR (qPCR) methods. We combined MNAzyme components with PCR primers and standard qPCR reagents to perform MNAzyme qPCR and reverse-transcription qPCR (RT-qPCR) assays with a set of universal reporter probes. Assays were performed on single targets and in multiplex formats that combined up to 5 different targets in a single reaction. A comparison of 3 targets amplified in single and triplex formats showed no significant differences with respect to detection limit or amplification efficiency. Likewise, we successfully converted single-target assays for 11 transcripts of interest to triplex assays containing 2 reference transcripts without having to optimize or modify the conditions. A quintuplex RT-qPCR that simultaneously quantified 5 transcripts with 5 universal probes produced high amplification efficiencies and r(2) values for all transcripts. Despite the large numbers of oligonucleotides in the reactions, we observed no false-positive signals, owing to the requirement of 4 target-specific binding events to produce a signal. A quadruplex assay that combined MNAzymes with methylation-specific PCR to measure epigenetic biomarkers of prostate cancer was capable of detecting a single methylated DNA allele in a background of 1000-10 000 unmethylated alleles. The MNAzyme qPCR was compatible with a rapid-cycling protocol. MNAzymes offer a flexible and unique approach to qPCR that is specific, sensitive, and easily multiplexed. The universal nature of MNAzyme reporter probes removes the need for target-specific probes, thereby making the development of new assays easier and cheaper.