Continuous-flow thermal gradient PCR Niel Crews & Carl Wittwer & Bruce Gale # Springer Science + Business Media, LLC 2007 Abstract 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 dem- onstration 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 straightfor- ward 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. Keywords PCR . Rapid cycling . Continuous-flow . Thermal gradient . Microfluidics 1 Introduction Continuous-flow polymerase chain reaction (CF-PCR) is an amplification technique in which a single fluidic channel is heated with spatial temperature variations such that a flowing sample experiences the thermal cycling required to induce amplification. This heating method reduces the thermal load to only that of the sample being amplified. By excluding the substrate from the thermal cycling, lower energy consumption and faster cycling can be achieved. This has been demonstrated with a variety of thermocycling techniques, including infrared (IR) heated PCR systems (Roper et al. 2007), shuttle PCR devices (Chiou et al. 2001), and CF-PCR instrumentation. CF-PCR was first dem- onstrated in a microfluidic device by Kopp and coworkers (Kopp et al. 1998). This foundational design consisted of a microfluidic serpentine channel embedded within a glass substrate. Three heaters were fixed to the chip to produce distinct thermal zones through which the fluid would pass. Other researchers have continued to improve the operation of this original 20-cycle device. Li and coworkers (Li et al. 2006) built a device whose 20-cycle serpentine micro- channel was narrower in the regions between the three temperature zones, thus reducing the inter-temperature transition time. Schneegass and coworkers (Schneegass et al. 2001) built a 25-cycle device from silicon and glass. The device included integrated heaters and temperature sensors which were fabricated on-chip using IC manufac- turing technology. Fukuba and coworkers (Fukuba et al. 2004) were able to automate the operation of a 30-cycle device using miniature pumps and valves. Sun and co- workers (Sun et al. 2002) have developed a 30-cycle CF- PCR device with integrated ITO heaters (indium tin oxide), thus making the device optically transparent. Obeid and coworkers (Obeid et al. 2003b) presented a device capable of the reverse transcription of RNA prior to its amplification in a 40-cycle serpentine channel (RT-PCR). The device was Biomed Microdevices DOI 10.1007/s10544-007-9124-9 N. Crews : B. Gale (*) Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA e-mail: bruce.gale@utah.edu C. Wittwer Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA