Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm ( N = 6) or preset cycling conditions ( N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.