Type of Document Master's Thesis Author Chen, Pin-Chuan Author's Email Address firstname.lastname@example.org URN etd-06152006-154657 Title Accelerating Micro-Scale PCR (Polymerase Chain Reactor) for Modular Lab-on-a-Chip System Degree Master of Science in Mechanical Engineering (M.S.M.E.) Department Mechanical Engineering Advisory Committee
Advisor Name Title Michael C. Murphy Committee Chair Dimitris E. Nikitopoulos Committee Member Ram Devireddy Committee Member Steven A. Soper Committee Member Keywords
- micro pcr
Date of Defense 2005-11-16 Availability unrestricted AbstractThe polymerase chain reaction (PCR) is a powerful technique used to exponentially amplify specific DNA sequences of interest through repetitive temperature cycling. The temperatures used in PCR are typically 90°C - 94°C for denaturation, 50°C - 70°C for renaturation, and 70°C - 75°C for extension. The double-stranded helix of nucleotides, carrying the genetic information, is separated during denaturation, reacts with chemical primers during renaturation, and becomes a complete, replica, double-stranded DNA helix structure during extension. The primary drawback of current commercial benchtop PCR machine is its cycle time due to its large thermal capacitance, and micro PCR is under developing using microfabrication technology; smaller and faster in different types of PCR are the primary goal in this study.
Continuous flow PCR is one of the primary types of micro PCR and it relies on a continuous flow through three temperature zones to achieve rapid thermal cyclings. To understand its biological performance, an experiment was carried out to study its limiting dynamic performance at different flow velocities from 1 mm/s to 15 mm/s and a thermal and fluidic numerical simulation was realized to give ideas of thermal performance and explain the experimental results. A 5.2 s/cycle for 500 bp and a 9.7 s/cycle for 997 bp were accomplished in this experiment. Thermal management was critical in PCR since the biological performance was primarily dependent on precise temperature control. Liquid crystal is a common tool in investigating thermal performance using its optical properties, which will have different color based on its local temperature. A liquid crystal was realized on renaturation zone of CFPCR chip and post image process was used to help interpret the experimental results. A non-uniform temperature distribution was observed due to the low thermal conductivity of substrate, polycarbonate, and non-uniform temperature supply.
Another goal in this research was to develop another type of PCR using different driving mechanism to search other possibilities and compare results with CFPCR. An electrokineitc shuttle PCR was developed and its basic idea was using electrokinetic force to drive DNA fragment through three static temperature zones in a single microchannel. The flow velocities realized in this experiment were 1mm/s to 3mm/s and lower amplification yields were observed. The reasons were unintentional flows such as siphoning flow and hydrodynamic flow, which made the DNA fragment move in a chaotic temperature sequences and result in a lower yield.
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