Microfabricated Device for DNA and RNA Amplification by Continuous-Flow Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction with Cycle Number Selection

Abstract

We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

Rapid PCR in a continuous flow device

“…15 Alternatively, DNA amplification can be achieved in a microfluidic platform by moving a PCR mixture in a microchannel repetitively through different temperature zones using a continuous-flow (CF) format. [24][25][26][27][28][29][30][31][32][33][34][35][36] The CFPCR approach can be conducted at relatively high speeds since it is not necessary to heat and cool the amplification chamber repeatedly. 26 Kopp et al were able to produce a relatively short target fragment (176 bp) in a cycle time of 4.5 s by starting the PCR with a large number of templates (y10 8 copies of a 1 kbp PCR product) along with nested primer sets.…”

Instrumentation of a PLC-Regulated Temperature Cycler with a PID Control Unit and Its Use for Miniaturized PCR Systems with Reduced Volumes of Aqueous Sample Droplets Isolated in Oil Phase in a Microwell

“…Among the DNA assay chips, a number of miniaturized instruments have been developed for polymerase chain reactions (PCR) because the PCR amplification is widely used as a molecular biological tool to replicate millions of copies of target DNA fragments by cycling through two or three temperature steps (denaturation, annealing and extension). [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] The miniaturization of PCR devices can take advantage of reduced consumption of biological sample necessary for PCR and of increased portability. In addition, the decreased cost of fabrication with a choice of polymer materials as a substrate for PCR reaction vessel allows one-time use of the chips, leading to elimination of false positive data resulting from carryover crosscontamination.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] In the continuous-flow format, DNA amplification can be achieved by shuttling a PCR cocktail in a microchannel repetitively through different isothermal zones. [14][15][16][17][18][19] The batch format chip is more suitable for miniaturization and high throughput operation; however, special care must be taken in the thermal management of a stationary PCR mixture to acquire quick transitions having low overshoots, together with good rejection of the disturbance.…”

“…10,15,17,[20][21][22][23][24][25][26][27] A personal computer running LabVIEW and GPIB controlled instrumentation has been developed by some research groups to build a home-made PID-controlled temperature cycling system. 10,[20][21][22] The graphical programming language used in LabVIEW is intended for automating and measuring equipment in a laboratory setup and allows even a non-programmer to build programs by simply connecting virtual representations of lab equipment on a screen.…”

Microfabricated valveless devices for thermal bioreactions based on diffusion-limited evaporation

“…There are two major types of miniaturized bioreaction systems: batch-based systems where the stationary reaction solution is heated or thermocycled inside a reaction chamber by either external heaters [3][4][5][6][7][8][9][10][11][12][13][14][15][16] or integrated on-chip heaters, [17][18][19][20][21][22][23][24] and continuous flow-based systems where the sample flows through certain temperature zones with well-defined flow rates. [25][26][27][28][29][30] Other novel approaches, such as on-chip rotary reaction, 31 noncontact infrared-mediated reaction, [32][33][34] electrokinetically synchronized reaction, 35 electrowetting-based reaction 36 and Rayleigh-Bénard convection-based reaction 37,38 have also been reported. Recent trends in miniaturized bioreaction systems are to integrate bioreactions with sample preparation, fluidic handling, and product detection to produce systems that can rapidly, conveniently, and economically extract information from raw biological samples with greatly reduced cost.…”