Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater

Abstract

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.

Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

“…Diagnostic applications include microRNA profiling, gene expression analysis, pathogen quantification, and cancer diagnostics (2,3 ). There is an ongoing development of novel devices on the basis of capillary systems, microfluidics, point-of-care testing, microbeads, and related technologies (6,(7)(8)(9)(10)(11)(12).…”

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

“…Therefore, without heating and cooling the container repeatedly, reagents circulate spontaneously and then experience three amplification processes at corresponding temperature zones to amply millions of target DNA copy numbers within one hour. Afterward, our research group established a convective capillary PCR (ccPCR) technique [11]. For the purpose of the convenience of liquid handling, the glass capillary tube as a reagent container was used in this platform.…”

Convective PCR Thermocycling with Smartphone-Based Detection: A Versatile Platform for Rapid, Inexpensive, and Robust Mobile Diagnostics

“…3.1d, e). More recently, capillary convective PCR platforms have been introduced that impose vertical thermal gradients via heating the side walls of thin capillary tubes containing the PCR reagent mixture [18][19][20] (Fig. 3.1f).…”

“…Closed-loop convective systems have also been demonstrated that transport reagents between PCR temperature zones in (d) oval [8] and (e) triangular [14] flow loops. (f) Thermal convection in capillary tubes heated from below has also been harnessed to perform PCR thermocycling [18]. Panel (a) reprinted with permission from Ref.…”