Date of Award:

5-2024

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biological Engineering

Committee Chair(s)

Keith Roper

Committee

Keith Roper

Committee

Foster Agblevor

Committee

James Powell

Abstract

Real-time quantitative polymerase chain reaction fluorescent curves are influenced by the quantification method, template length, primer sequence, polymerase activity, reaction conditions, and unwanted side reactions. Interpretation of these curves depends on understanding the subjacent mechanisms by which those factors can alter the curve profile, particularly on highly diluted analytes or impure samples. However, such factors are individually described today, or their interplay is not fully developed. The present work examines the effect of primer dimer formation and extension, resource competition, template reannealing, and polymerase thermodegradation, in addition to novel descriptions of polymerase competitive inhibition, extended primer amplification, and the probability of complete extension, which had not been included in previous models. The model is able to reproduce experimental curves across variations of fluorescence method, template length, master mix content, and cycle durations. These new approaches enable a mathematical description of phenomena such as lower plateau outcomes at highly diluted templates, competitive polymerase inhibition, and incrementing the quantification cycle in long templates. This kinetic model improves the capabilities to design real-time quantitative polymerase chain reaction methods and interpret curves, particularly from challenging scenarios and new applications.

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