Figure 1. Spike in dilutaion curve comparing ddPCR with superRCA.
Taqman probe has been the fundamental element for the allele distinction function in various PCR based mutation detection technologies. In emulsion/microcompartment digital PCR (dPCR) and quantitative PCR (qPCR), the Taqman probe distinguishes different alleles during PCR amplification steps. However, the hydrolysis feature in the Taqman probe relies on the 5’ to 3’ exonuclease possessed by the PCR polymerases, thus limits the selection of PCR polymerase to Taq DNA poly which is suboptimal in PCR replication fidelity.
Other technologies for allele distinction such as BEAMing assays employs a bead-based amplification which allows to use high fidelity DNA polymerase to generate enough target molecules in bead and perform Taqman probe based allele distinction after the PCR amplification. But it is quite tricky to ensure (one input molecule + bead) per emulsion to generate single molecule derived bead colony resulting excessive number of empty beads during enumeration.
All Taqman probe-based allele distinction relies on the hybridization affinity difference between the match/unmatch target so that only the matched probe give rise to fluorescent signals while the unmatched will fall off. However, the Gibbs free energy difference (ΔG) attributed by the single nucleotide (SNP distinction) heavily influenced by the environmental sequences (the sequence that Taqman recognizes), which determines the severity of missing binding of the Taqman probe.
In superRCA technology-based mutation detection assays, we use padlock probe based allele distinction mechanism. The Padlock probe utilize four interdependent mechanism to improve the genotyping sensitivity: 1. Probe hybridization (binding to the matched target), 2. Ligase mediated allele distinction (only perfect match base pair can be ligated into replicable circles). 3. Circle confirmation (only successfully ligated probe can contribute to a 2nd rolling and yield fluorescent signal while the miss matched probe contributes nothing to the total fluorescence), 4. Major voting mechanism (the concatemer based padlock probing employs hundreds to thousands of padlock probe to sense the same target molecule).
These four mechanisms enable extreme high fidelity allele distinction that is used in the superRCA based assays to provide cutting edge sensitivity.