Thirty-Cycle Temperature Optimization of a Closed-Cycle Capillary PCR Machine

Abstract

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-µL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oilfilled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94°C-96°C, which is identical to commercial machines. The optimal extension temperature of 62°C-66°C is lower than reported for Taq DNA polymerase (70°C-80°C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 min for 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

“…The main reason for doing it is that the amplification length of each target gene fragment in the present microfluidic system is relatively small, and therefore the time it takes for the solution to flow through the extension zone may be enough to induce the multiplex extension reaction. In addition, it is possible that the primer extension reaction occurs when the reaction solution is inside of the annealing heat zone and between the annealing and extension heat zones (annealing takes places as the reaction solution travels between heat zones) (Chiou et al 2001(Chiou et al , 2002Chen et al 2007b). Unless Fig.…”

“…Among them, the oscillatoryflow PCR devices have obtained increasing attention in recent years, due to their salient features such as cycle number/dwell time flexibility, ease of real-time detection implementation, large footprint reduction, and ability to process multiple samples in parallel. In 2001, Chiou et al reported a close-cycle capillary PCR thermocycling machine, where the bidirectional pressure-driven flow and in situ optical position sensors have been integrated (Chiou et al 2001(Chiou et al , 2002. Using this device, 30 cycles of a 500-bp product were performed in 23 min with 78% amplification efficiency.…”

“…The heaters used for the oscillatoryflow PCR have changed from the metal/ceramic blocks with a certain heating cartridge (Chiou et al 2001(Chiou et al , 2002Auroux et al 2003a;Hardt et al 2004;Münchow et al 2005;Frey et al 2007;Sugumar et al 2010) to the Peltier heater (Chen et al 2007b;Paik et al 2007) and the film heaters integrated on the chips (for example indium tin oxide (ITO) (Cheng et al 2005), platinum (Bu et al 2003;Wang et al 2005;Polini et al 2010;Sciancalepore et al 2011), chromium/aurum (Ugsornrat et al 2008), and others (Baker et al 2003;Auroux et al 2003bAuroux et al , 2004aSista et al 2008)). And, the temperature sensor elements include the commercially available thermocouples (Chiou et al 2001(Chiou et al , 2002Auroux et al 2003a;Chen et al 2007b;Paik et al 2007), the thermistors (Sista et al 2008;Sugumar et al 2010), and the on-chip film sensors (Bu et al 2003;Wang et al 2005;Polini et al 2010;Sciancalepore et al 2011). The use of integrated film heater and sensor can increase the degree of integration of the microfluidic device, although this may result in an increased cost of fabrication and may require special equipment and increased time for fabrication.…”

Reduction of water evaporation in polymerase chain reaction microfluidic devices based on oscillating-flow

“…3 Methods relying on passivation agents directly added to the amplification mix generally require optimal relative concentrations of surface modifiers, reagents, and DNA, and potentially fail when used in microchannels of different volumes. To overcome Taq adsorption on the sidewalls of their oscillatory capillary PCR machine, Chiou et al 38,39 reported on the use of high concentrations of Taq DNA polymerase ͑0.18− 0.7 unit/ l͒, flowing into a polytetrafluoroethylene microcapillary over 30 cycles. Here, a static silanization of glass is performed prior to the amplification experiments, allowing us to work with a standard quantity of Taq polymerase ͑0.02 unit/ l͒.…”

“…Here, a static silanization of glass is performed prior to the amplification experiments, allowing us to work with a standard quantity of Taq polymerase ͑0.02 unit/ l͒. The PCR mixture also contains the biological detergent Tween-20; however, different from previous studies, 38,39 we could not note relevant stability issues for our droplets, which is likely related to the low fluid velocity ͑3 ϫ 10 −3 m / s͒. Glass capillaries treated by the coating solution are tested in the microfluidic devices using the same thermal profile.…”