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

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Previous PCR cycle control methods used optically labeled L-DNA enantiomers to determine thermal cycling switch points.
• These methods assume L-DNA hybridization accurately reflects D-DNA primer and target hybridization kinetics.
• Factors affecting hybridization kinetics can impact PCR accuracy and lead to delayed or failed amplification.

Purpose of the Study:

• To determine the optimal L-DNA concentration and strand ratios for accurate mirroring of D-DNA hybridization using the Van't Hoff equation.
• To evaluate the performance of an L-DNA control algorithm in compensating for hybridization errors in real-world PCR applications, such as SARS-CoV-2 detection.
• To assess the L-DNA control algorithm's ability to prevent false negatives and delays in PCR cycle quantification (Cq) under challenging sample conditions.

Main Methods:

• Applied the Van't Hoff equation to analyze simultaneous fluorescence and temperature measurements during L-DNA controlled cycling.
• Compared optical and thermal switch points as a function of varying total L-DNA concentration and strand ratios.
• Tested the L-DNA control algorithm on the SARS-CoV-2 N2 reaction using simulated 'unextracted' samples with nasopharyngeal and high salt backgrounds.

Main Results:

• Optimal L-DNA concentration equals the initial D-DNA primer concentration; excess of one L-DNA strand over the other best mimics D-DNA behavior.
• The L-DNA control algorithm prevented significant Cq delays in simulated unextracted SARS-CoV-2 samples, unlike preset cycling conditions.
• Preset cycling conditions showed significantly increased Cq values in the presence of nasopharyngeal or high salt backgrounds.

Conclusions:

• The L-DNA control algorithm effectively adapts PCR cycling conditions to compensate for hybridization errors.
• This adaptive control minimizes the impact of sample matrices and inhibitors, preventing false negatives and delayed Cq values.
• L-DNA based control offers a robust solution for improving PCR reliability, particularly in diagnostic applications without prior nucleic acid extraction.