Applied & Computational Mathematics seminar: Removing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR

Speaker:  Rob De Jaco, National Institute of Standards and Technology

Title:  Removing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR

Abstract:  For many years, DNA amplification by polymerase chain reaction (PCR) has been modeled as a stochastic branching process. However, such models do not distinguish between complementary strands of DNA and have not yet been linked to the fluorescence measured in real-time PCR. As such, they cannot satisfy the initial conditions encountered in assays starting from RNA, they do not provide a realistic description of the fluorescence reported by common probes, and they have not been used to quantify the uncertainty in the fluorescence measured. To address these issues, a new branching process accounting for DNA complementarity is presented. Analysis of the expected value identifies an additional time scale that occurs at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification. Linking the branching process to the fluorescence measured by an analog of Beer’s Law allows for an objective and a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to an analytical expression between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the initial copy number and its uncertainty are quantified from fluorescence measurements in the regime of exponential growth. 

Time:  Friday, October 21, 2022, 1:30pm-2:30pm

Place:  Exploratory Hall, Room 4106 or Zoom