L-DNA calibrators for PCR amplicon characterization.

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Real-time polymerase chain reaction (PCR) is a powerful molecular biology technique.
• Post-PCR methods like melt analysis and target quantification enhance PCR utility.
• L-DNA stereoisomers have previously improved PCR sensitivity and specificity.

Purpose of the Study:

• To utilize L-DNA sequences as internal calibrators for confirming PCR amplicon specificity.
• To estimate initial target concentration using L-DNA calibrators.
• To characterize PCR amplicons within a single sample.

Main Methods:

• Real-time PCR and melt analysis were performed using an *S. mitis* reaction.
• L-DNA sequences served as internal calibrators with known melt temperatures.
• Melt curves were analyzed to generate melt signatures compared to L-DNA calibrators.
• Initial target concentration was estimated using a multi-step method involving peak amplitude extraction, intercalator fluorescence conversion, and sigmoidal modeling of efficiency decay.

Main Results:

• L-DNA calibrators enabled the confirmation of amplicon specificity and estimation of initial target concentration.
• The *S. mitis* amplicon melt signature was consistent (0.8143 ± 0.0058), translating to 84.96 ± 0.10 °C.
• Initial target concentration estimates were accurate above 10^2 copies/µL, with coefficients of variation of 120% at 10^2 and 22% at higher concentrations.

Conclusions:

• L-DNA strands can function as internal calibrators in real-time PCR.
• This method allows for single-sample characterization of PCR amplicons.
• The approach enhances the reliability and quantitative accuracy of PCR assays.