2Department of Materials Engineering, National Pingtung University of Science and Technology, Pingtung, Taiwan
3Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan
4Graduate Institute of Clinical Medicine, National Taiwan University, Taipei, Taiwan
Herein we describe a simple platform for rapid DNA amplification using convection. Capillary convective PCR (CCPCR) heats the bottom of a capillary tube using a dry bath maintained at a fixed temperature of 95°C. The tube is then cooled by the surrounding air, creating a temperature gradient in which a sample can undergo PCR amplification by natural convection through reagent circulation. We demonstrate that altering the melting temperature of the primers relative to the lowest temperature in the tube affects amplification efficiency; adjusting the denaturation temperature of the amplicon relative to the highest temperature in the tube affects maximum amplicon size, with amplicon lengths of ≤500 bp possible. Based on these criteria, we successfully amplified DNA sequences from three different viral genomes in 30 min using CCPCR, with a sensitivity of ~30 copies per reaction.
PCR is the primary technique used for amplifying specific nucleic acid sequences (1). Traditional PCR requires the use of a delicate thermal controller to repeatedly heat and cool samples, typically for 35–50 cycles. During their early development, the size, cost, and long ramping time of traditional PCR thermocyclers limited their use to major hospitals and laboratories. Recently, new platforms have been developed that simplify the equipment and shorten amplification times. One approach increases the ramp rates of temperature changes through the use of a Peltier element with high electric power (2-5), shortening the time for PCR amplification from 2–3 h to 30–40 min. However, such increases in ramp rates require a more delicate and costly thermal controller than traditional thermocyclers with slower ramp rates. Another approach is to replace the thermal cycler altogether with a specially designed microfluidic chip. This platform circulates reagents inside microchannels through three zones at different fixed temperatures (6-10). The microelectromechanical systems (MEMS) approach for fabricating biochips can shrink the size of such systems dramatically. However, such systems require external pumping sources, complex microchannel design, and two or three temperature controllers running at the same time.
Natural convection induced by density changes of fluids within temperature gradients can be also applied to DNA amplification (11). When the reagents in a sample circulate repeatedly through different temperature zones driven by natural convection, it is possible for the sample to undergo the three steps of a PCR cycle—nucleic acid denaturation, annealing, and extension—in a single circulation. Convective PCR can reduce the amplification time from multiple hours to 30–40 min (12,13). Using this platform, a dedicated thermal cycler— the most expensive component of conventional PCR—is not needed. However, current convective PCR systems use two or more independent temperature controllers and complicated designs requiring a specific shape of tubing or additional chambers to circulate fluids completely (14-18). Moreover, skillful manipulation is necessary for certain operations such as loading and unloading reagents from thin tubing and sealing both ends of the tube without trapping air bubbles inside. Every step is crucial for successful operation. Furthermore, very few studies have examined the differences in primer and amplicon design between traditional and convective PCR, limiting the versatile application of convective PCR.
Here we describe a new capillary convective PCR platform (CCPCR) where the capillary tube is mounted on a heater with a fixed temperature at 95°C. No complex microfluidic chip design, special tubing or vessels are necessary. The continuously heated capillary base drives the lowest part of the sample to rise by convection while at the same time denaturing the template. As this portion of the sample rises, its temperature falls due to cooling from the surrounding air. When the sample reaches the cool temperature zone near the top of the tube, it undergoes annealing and extension following which the DNA template sinks and is heated again. In this way, PCR cycles are achieved by natural convection.
In such a simple platform, we demonstrate that correct primer and amplicon design are essential for successful DNA amplification. Because of the simplicity of CCPCR, nucleic acid amplification can be accomplished without costly and delicate thermocyclers and without any additional complicated hardware. We believe that this platform will offer a new DNA amplification method with an ease of operation and low cost that is well suited for point-of-care applications in third-world countries.Materials and methods Setup configuration of capillary convective PCR
CCPCR employs a standard dry heat bath with temperature feedback control as the heat source (Supplementary Figure S1). The sample container is a commercial glass capillary tube with a closed bottom (LightCycler Capillaries 100 µL, Roche). Each is 51 mm long and the inner and outer diameter of the capillary tube is 2.3 mm and 3.2 mm, respectively. Two handmade heating blocks with different drill hole depths (4 mm and 30 mm) are placed in a dry bath maintained at 95°C (Digital dry bath incubator, Cat. no. BL-3002, Violet BioScience Inc., Taipei, Taiwan). The first step is to place the glass capillary tubes containing the samples in the heating block with 30 mm–deep holes. The whole tube is heated entirely by conduction within 10 min to activate the Taq DNA polymerase. After that, the tubes are transferred to the block with 4 mm–deep holes and supported with a plastic capillary holder stand. The tubes are heated only at the bottom to create natural convection for DNA amplification in 30 min.