The Science Behind Single-Copy Cancer DNA Detection

Bicycle PCR contains two sets of thermocycling (bi-cycle) and a blocker. The blocker prevents normal DNA replication in the first set of thermocycling. The blocker does not prevent normal DNA replication in the second set of thermocycling.

Bicycle PCR improves accuracy by enriching the percentage of cancer DNA above the error rate of the downstream step. For example, Sanger sequencing has an error/noise of 5%, which means a sample containing 0.01% cancer DNA cannot be reliably detected. Bicycle PCR enriches a sample containing 0.01% cancer DNA, well above 5% during the PCR step. This allows accurate detection of cancer DNA.

The secret sauce was to lower the concentration of the blocker, which makes an unnecessary weakness.

Blocking of normal DNA occurs when the blocker hybridizes before the primer. All DNA is replicated when the primer hybridizes before the blocker. Bicycle PCR leverages temperature and time to control whether the blocker or the primer hybridizes first.

Previous blocking methods demonstrated that if the blocker concentration was reduced, the blocking was no longer effective, even though the blocker was still present. Additionally, COLD-PCR demonstrated that a blocker at low concentrations could still effectively block if a blocker annealing step occurred prior to the primer annealing step. By combining these two well-documented scientific principles, Bicycle PCR allows for the modulation of blocking effectiveness based on the presence/absence of a blocker annealing step prior to the primer annealing step.