A thermally baffled device for highly stabilized convective PCR

Abstract

Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field.

Figure 1

Design of the thermally baffled device and the temperature profile inside the capillary tube. (A) The thermally baffled device was designed to reduce the influence of the environment. (B) Thermo-image analysis showing the temperature profile with a bottom-heating apparatus only, (C) same bottom-heating apparatus covered with polycarbonate, or (D) a thermally baffled device. The thermo-image was observed using an infrared thermometer. The area of the thermal baffle is indicated by orange lines and arrows. The temperature was measured at 18.5 mm from the bottom of the tube (black arrowhead) and analyzed by a ThermoData Analysis System. The temperature profiles of the above three models under three different settings (28°C, black line; 38°C, red line; and 28°C with wind, blue line) were also compared. The temperature profile was obtained for three independent experiments with similar results.

Figure 2

YHV-specific amplicon was amplified by conventional PCR and convective PCR using the thermally baffled device. (A) Different numbers of copies (10³–10¹) of a plasmid containing a YHV-specific sequence as the template were amplified by conventional PCR or convective PCR with the thermally baffled device at 28°C, (B) 38°C, 45°C or under stable air-flow conditions (1 m/s). The products were analyzed on a 12% polyacrylamide gel in 1 × TAE buffer. M, DNA molecular weight marker (bp); R, reaction; N, no template control; P, positive control (at 28°C). PCR amplification was performed in at least three independent experiments with similar results.