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Analysis of rapid thermocycling for the polymerase chain reaction
PCR (polymerase chain reaction) has become an important diagnostic tool for the identification of pathogens. There has been a dramatic increase in usage of PCR since the late eighties and it is expected that future use will increase even more. PCR devices continue to evolve and performance should increase significantly at reduced cost. This study focuses on the principles of rapid PCR with the PCRJet. The PCRJet is a new thermocycler patented by Megabase Research Products (Lincoln, NE). Performance of the PCRJet is thoroughly analyzed-including (1) an engineering approach to study the PCRJet's performance and (2) a biochemical approach to amplification of a number of different DNA fragments. This PCRJet study is necessary to develop a rapid and portable Ranque-Hilsch based PCRJet machine in a joint project between Megabase Research Products and the University of Nebraska. ^ More than 600 PCR experiments have been performed in several versions of the PCRJet machine. The fastest PCR accomplished to date has been for an 85 base pair DNA fragment; thirty cycles for this amplicon required only 78 sec. The longest fragment the PCRJet has amplified has been 2331 base pairs; thirty cycles in the PCRJet took 10.3 min . The PCRJet speed compares favorably to commercially available devices which require thirty minutes to three hours to complete thirty PCR cycles. ^ The PCRJet speed originates in its heat transfer by convection using high velocity gasses. In PCR, a small volume of an aqueous solution containing the template DNA, primers, polymerase, and other chemicals is cycled through a defined, sequential temperature protocol. Of the three modes of heat transfer, thermal conduction has been the choice for most devices to attain the temperature protocol. Consequently, most of the time in a thermal block PCR cycle is spent non-productively in transition between DNA denaturation, primer annealing, and enzymatic elongation temperatures. Aspects of the convective heat transfer utilized by the PCRJet are analyzed within this dissertation. Some computational fluid dynamics results of the PCRJet heat transfer are shown. ^
Chemistry, Biochemistry|Engineering, Chemical
Whitney, Scott E, "Analysis of rapid thermocycling for the polymerase chain reaction" (2004). ETD collection for University of Nebraska - Lincoln. AAI3131568.