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

Citations Over TimeTop 1% of 2002 papers

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 μm, 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 × 106−1.6 × 108 and 6.2 × 106−2 × 108, respectively. Successful amplification of a single-copy gene (β-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-μL 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-μL 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.

Related Papers

• → The influence of quality of primers on the formation of primer dimers in PCR(2020)27 cited
• → Isolation and Characterization of Plant Genomic DNA Sequences via (Inverse) PCR Amplification(2003)14 cited
• → A repeat protein‐based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR(2016)3 cited
• → Detection of Specific DNA Sequences in Environmental Samples Via Polymerase Chain Reaction(1994)10 cited
• → The Polymerase Chain Reaction(2014)