(174ap) Quantitative Analysis of DNA Helicity State within a Convective PCR Chamber through CFD Simulations

Through the polymerase chain reaction (PCR) cycles of denaturation, annealing, and extension, each of which optimally occurs at specific temperature zones, nucleic acids can be replicated to a billion-fold. Thus, PCR is of significant importance in sensitive nucleic acid-based applications, especially DNA amplification. In conventional PCR, repeated heating and cooling of the PCR reagents through the involved steps at their respective temperatures and during precise times is vital to ensure an efficient process. Hence, conventional PCR is accompanied by limitations for the implementation of rapid and portable diagnosis. In this regard, convective PCR (cPCR) has emerged as a rapid and portable PCR approach, establishing micro-scale thermal convection inside a cylindrical PCR chamber where only two fixed temperatures are required (top ~58 °C and bottom ~96 °C). Thus, reagents in cPCR are continuously circulated through denaturation, annealing, and extension temperature zones, which are simultaneously established in different regions within the chamber. As a result, during the thermocycling processes in cPCR, DNA helicity state, that is, the percentage of double stranded DNA (dsDNA) that is in a double helix state, would spatially vary within the chamber. dsDNA at the low temperature top is thus expected to be ~100% helicity (fully dsDNA), while it drops to ~0% helicity as the temperature increases and the dsDNA melts into single stranded DNA (ssDNA) at the bottom of the chamber. Herein, we present a quantitative analysis of DNA helicity state within a cPCR chamber through computational fluid dynamics (CFD) simulations. To this end, in the first step, the temperature distribution and concentration of the DNA species are obtained via a coupled flow and reaction model in a CFD simulation software package (STAR-CCM+). The second step involves the utilization of the web-based application (uMelt) to determine the helicity percentage of a DNA sequence with a specific GC content and base pair length as a function of temperature. Accordingly, by the temperature field obtained in CFD simulations, the corresponding DNA state for each point can be determined. The results of this study will be key to the conduction of melting curve analysis in cPCR systems in the future, which could potentially allow us to skip post-processing steps, like gel electrophoresis, to determine the quality of the reaction products, making PCR cheaper and more versatile.