Reducing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR.

Area of Science:

• Molecular Biology
• Biophysics
• Biostatistics

Background:

• Real-time Polymerase Chain Reaction (PCR) assays measure nucleic acid amplification through fluorescence.
• Current methods may have bias and lack robust uncertainty quantification.
• Accurate quantification is crucial for diagnostics and research.

Purpose of the Study:

• To develop a new approach for relating nucleic acid concentration to fluorescence in real-time PCR.
• To reduce bias and quantify uncertainty in fluorescence measurements.
• To enable uncertainty quantification in PCR (UQ-PCR).

Main Methods:

• Coupling a two-type branching process for PCR with a fluorescence analog of Beer's Law.
• Distinguishing between complementary DNA strands for stoichiometric reaction descriptions.
• Analyzing expected copy-number and variance to identify amplification dynamics and sources of error.

Main Results:

• The approach provides a stoichiometric description of probe-DNA reactions and captures initial RNA assay conditions.
• Identified dynamics at low cycle numbers and quantified contributions from volume transfer, amplification imperfections, and strand-specific synthesis.
• Established analytical relationships between amplification efficiency and limit of detection through uncertainty quantification.

Conclusions:

• The developed method reduces bias and quantifies fluorescence uncertainty in real-time PCR.
• This framework enables a priori background fluorescence description and improved limit of detection analysis.
• The work paves the way for UQ-PCR, quantifying both input copy-number and its uncertainty from fluorescence kinetics.