Continuous-flow thermal gradient PCR
Continuous-flow thermal gradient PCR is a new DNA amplification technique that is characterized by periodic temperature ramping with no cyclic hold times. The device reported in this article represents the first demonstration of hold-less thermocycling within continuous-flow PCR microfluidics. This is also the first design in which continuous-flow PCR is performed within a single steady-state temperature zone. This allows for straightforward miniaturization of the channel footprint, shown in this device which has a cycle length of just 2.1 cm. With a linear thermal gradient established across the glass device, the heating and cooling ramp rates are dictated by the fluid velocity relative to the temperature gradient. Local channel orientation and cross-sectional area regulate this velocity. Thus, rapid thermocycling occurs while the PCR chip is maintained at steady state temperatures and flow rates. Glass PCR chips (25 × 75 × 2 mm) of both 30 and 40 serpentine cycles have been fabricated, and were used to amplify a variety of targets, including a 181-bp segment of a viral phage DNA (ΦX174) and a 108-bp segment of the Y-chromosome, amplified from human genomic DNA. With this unique combination of hold-less cycling and gradient temperature ramping, a 40-cycle PCR requires less than 9 min, with the resulting amplicon having high yield and specificity.
KeywordsPCR Rapid cycling Continuous-flow Thermal gradient Microfluidics
Authors acknowledge the Utah State Center of Excellence Grants, the University of Utah Synergy Program, and the NSF IGERT Program for the funding of this work. Authors also wish to thank the respective members of the Wittwer and Gale research labs, whose valued contributions have allowed for this project to advance at a welcome pace.
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