Continuous-flow thermal gradient PCR

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 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 x 75 x 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 (PhiX174) 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.