pHBV-48 (19), HCV plasmid (20), and HIV-1 vector pNL4–3 (21), were used as template DNAs for amplification using traditional and capillary convective PCR. All primer sets (Supplementary Table S1) were designed by software (LightCycler Probe Design Software 2.0, Roche) and synthesized by Mission Biotech, Taiwan.PCR amplification and electrophoresis
Each CCPCR reaction contained 3 µL DNA template, 7.5 µL LightCycler FastStart DNA Master Hybridization Mixture (Taq DNA polymerase, PCR reaction buffer, 10 mM MgCl2, and dNTP mixture; Roche, Germany), 9 µL 25 mM MgCl2, 1.5 µL 10 µM each primer, and 52.5 µL double-distilled water. The samples in the capillary tubes for CCPCR were covered with 10 µL mineral oil (Cat. no. M5904, Sigma Aldrich, United States). The cycling conditions for the traditional thermal cycler (TGradient, Biometra, Germany) were: 95°C for 10 min (1 cycle); 40 cycles of 95°C for 20 s, 65°C for 20 s and 72°C for 30 s; then 72°C for 7 min. Two microliters of PCR or CCPCR products were run on a 2% agarose electrophoresis gel (Cat. no. MJ-105, Shorter Mini Gel System, Blossom Biotechnologies Inc., Taipei, Taiwan) containing 1µg/mL ethidium bromide submerged in 1×Tris-acetate–EDTA (TAE) buffer at 100 V for 40 min.Particle image velocimetry and temperature measurement
The flow pattern in a 75-µL volume capillary tube [h/d = 7.7 (h/d denotes ratio between the height of sample in the capillary tube and the inner diameter of capillary tube)] was visualized by particle image velocimetry (PIV) (TSI, St. Paul, MN). The working fluid was deionized (DI) water and the flows were seeded with 50-µm diameter polystyrene polyamide particles (ρp; 1005 kg/m3). After preheating for 10 min, the particles fluoresced at ~585 nm when excited with an Nd:Yag laser (λ = 532 nm); images were captured on a charge-coupled device (CCD) camera with a frame rate of 15 frames per second (fps) and overlapped. The particle-image fields were analyzed using an interrogation program written in MATLAB (MathWorks, Natick, MA, USA). The signal-to-noise ratio of spots on the cross images was improved with an arithmetic and geometric correlation function averaging process. The temperature of the interface (Tl) between the solution and oil was measured using a temperature recorder (PC-Based Data Acquisition Unit MX100; Yokogawa, Tokyo, Japan). The depth of thermocouple wire in tubes can be fixed at the interface using a custom-built steel holder with a special design for minimizing interference while the fluid is circulating.Melting curve analysis
Each sample contained 18 µL PCR or CCPCR products and 2 µL SYBR Green I (1:1000 dilutions from stocks; Invitrogen, Carlsbad, CA). The melting curve was analyzed by using a LightCycler 2.0 real-time PCR machine (Roche). The thermal conditions of the melting curve were: 95°C for 1 min, 60°C for 1 min, a temperature ramp of 0.1°C/s to 95°C in continuous fluorescence acquisition mode, and then 30°C for 1 min.Results and discussion
In our platform, the temperature gradient in the sample is created when the tube is heated from below and cooled by the surrounding air, causing natural convection to cycle the sample solution through the thermal gradient required for DNA amplification (Figure 1A). There are two challenges for the successful application of this simple platform: (i) to guarantee the creation and stability of the necessary flow and temperature fields for denaturation, annealing and extension when only one temperature control is available, and (ii) to design appropriate primers and amplicons suitable for use with the temperature gradients generated by the platform.
In traditional PCR, temperature settings and their duration are determined by amplicon length, GC content of the DNA template, and secondary structure (22). In contrast, convective PCR requires temperature gradients in a system generating flowing fields, so reagents continuously experience different temperatures during their circulation. Figure 1B (top) shows the typical changes in sample temperature during one circulation inside a capillary tube heated from below. The Th (the hottest temperature) zone is located at the bottom of the tube and the Tl (the lowest temperature) zone is located at the upper interface between the covering oil and reagents. To ensure that the three steps of a PCR cycle occur inside the capillary tube, the Td (denaturation temperature of the amplicon) should be lower than Th and Tm (melting temperature of the primer) should be higher than Tl. Obviously, the time for an effective reaction in the denaturing/annealing/extension steps depends on the temperature differences: Th–Td and Tm–Tl.