Studying On The Applicability Of Rayleigh Benard Convection Based Microfluidic Polymerase Chain Reaction System

PrefacePolymerase chain reaction(PCR) is a molecular biological method for in vitro amplification of nucleic acid molecule through repeated temperature cycling. The temperatures that are typically used in PCR include denaturation of double-strand DNA molecule, renaturation of the primers to the single-stranded DNA template, and enzymatic extension of the primers. The quantities of the molecule are doubled in one cycle, and in 30-40cycles it can be amplified nearly to 10~9 of the initial number. PCR can produce sufficient quantities of material for detection.In this conventional bench-top thermocyclers, cycling times are primarily limited by the rate at which inactive hardware elements(e.g., metal blocks) can be heated and cooled, rather than the kinetics of the reaction itself. Consequently, it is not uncommon for a 30-40 cycle amplification reaction to require 2-3 h of total time to complete, thereby limiting achievable throughput.For rapid and high-throughput analysis, it is particularly important to fabricate new type PCR device performing DNA amplification rapidly. The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems(MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Since the introduction of the first PCR chip, all kinds of PCR microfluidic technologies have facilitated DNA amplification with much faster rates as the result of smaller thermal capacity and larger heat transfer rate between the PCR sample and temperature-controlled components, which have advantages of small sizes, fast ramping rates, low cost and high integration and so on.So far, there are two categories of chip-based PCR, flow-through PCR and stationary chamber PCR, both of which have attracted great deal of interest and are exhibiting a rapid development. Recently, another convection-driven PCR microfluidic chip reported by Krishnan et al. In 2002(published on the magazine of Science), although only in its infancy, has been proven to be capable of rapid DNA amplification.In their work, Rayleigh Benard convection was used to perform PCR amplification(191bp-474bp) in a 35-μl cylindrical cavity in 40 minutes, and the temperature cycling was achieved as the flow continuously shuttles fluid vertically between the two temperature zones of annealing/extension(top, 61℃) and denaturation(below, 97℃). There is no need of an external force to drive the fluid through the different temperature zones. As compared with the flow-through or chamber stationary PCR microfluidics, this kind of convection-driven PCR microfluidics have the advantages of simplicity in design, cheapness of fabrication, and much faster temperature transition speed.In this study, based on the previous description of the convection microfabrication design, the polycarbonate(PC) is chosen as the reaction chamber material to perform the convection-driven PCR. After adjusting the size and insulation of the devices, the temperatures of the upper surface and passivating the inner surface of the chamber, rapid DNA amplification has been achieved in 20 minutes, much faster than that reported by Krishnan. Additionally, a lager scale of various sizes of DNA fragments, ranging from 100bp to 750bp, can be amplified successively on this device, which in turn enhances the application value of this device.Materials and MethodsPolycarbonate(PC) is selected as proper reaction chamber materials to perform DNA amplification, which neither inhibit the PCR reaction nor reduce the amplification efficiency, testified experimentally. The PCR products of plasm id DNA and phage DNA amplified by in a conventional thermocycler using a three-temperature cycling protocol are used as the template. To get more effective amplification, some alterations are carried out in this study, including the size of the cartridge and heat insulation of the reaction device, temperatures applied on the upper surface. BSA is also put in the PCR cocktail to prevent the absorption of the PCR mixture to the chamber. PCR products are identified by common gel electrophoresis.ResultsAn optimum temperature, 55℃at the upper surface and 99℃on the upper surface of the chamber was proved to be a proper reaction temperature condition. All PCR products of plasmid DNA and phage DNA generated in a conventional thermocycler using a three-temperature cycling protocol, used as the template, can be amplified in this micro-fabricated convectional PCR device. The lengths of the target DNA are 144bp, 258bp, 500bp and 750bp, respectively. But in parallel reactions for one time, different results were observed in different chambers; and for a certain chamber, different results were also observed in different experiments. The results of smaller size of the reaction cartridge, supplement of heat insulation treatment on the device, and adding BSA to the PCR cocktail are more satisfied.ConclusionA low-cost polymerase chain reaction device applying convectional principle has been successfully fabricated. After the optimization of the reaction condition, DNA amplification has been achieved in 20 minutes and a lager scale of various sizes of DNA fragments, ranging from 100bp to 750bp, can be amplified successively on this device, however, the reaction is sensitive to the temperature of the environment. It is hard to use it broadly; further study should be paid on it.