Reducing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR.

ABSTRACT: We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer's Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and str

SUBMITTER: DeJaco RF 

PROVIDER: S-EPMC10423706 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature