TY - GEN
T1 - A continuous flow polymerase chain reactor for DNA expression analysis
AU - Sirr, Noel
AU - Ciobanu, Doina
AU - Grimes, Ronan
AU - Davies, Mark
PY - 2006
Y1 - 2006
N2 - The polymerase chain reaction (PCR) has revolutionised molecular biology, and is at the forefront of many current efforts to document and understand human genetic diversity. Recent years has seen a move towards incorporating the PCR technique into a micro Total Analysis System (μTAS) thus exploiting its full potential. Micro scale PCR design offers the opportunity to integrate all functional steps of DNA expression analysis into a miniaturised device allowing for high throughput and reduced analysis times, reduced sample volume requirements and cost efficiency. Consequently, it is desirable to replace the traditional stationary or well based thermal cyclers with continuous flow designs. A continuous flow polymerase chain reaction device consisting of a cylindrical heating core, which is segmented axially into three symmetric heating zones providing the denaturating, annealing and extension phases of the polymerase chain reaction, and a flow through capillary tube which is wound helically around the core has been fabricated and shown to consistently amplify target plasmid DNA samples. At the inlet to the device, PCR samples are segmented into droplets and entrained in an immiscible carrier fluid to eliminate cross contamination between PCR samples. This approach also prevents degradation of the micro-reactor droplets from inhibitory effects posed by the high surface to volume ratios associated with the device. The droplet train is then cycled through the capillary tube with each complete revolution constituting one cycle of the PCR reaction. The results reported in this paper include, initial validation of the spiral cycler design in comparison to an existing commercial PCR platform, and subsequent optimisation of the reaction time and its effects on the devices performance. The spiral thermal cycler has demonstrated successful PCR amplification at the nano scale with stable trains of 30-35nl droplet volumes being processed in an amplification time of 32 minutes. At this level the device offers the potential to process approximately 3500 such droplets in of order one hour.
AB - The polymerase chain reaction (PCR) has revolutionised molecular biology, and is at the forefront of many current efforts to document and understand human genetic diversity. Recent years has seen a move towards incorporating the PCR technique into a micro Total Analysis System (μTAS) thus exploiting its full potential. Micro scale PCR design offers the opportunity to integrate all functional steps of DNA expression analysis into a miniaturised device allowing for high throughput and reduced analysis times, reduced sample volume requirements and cost efficiency. Consequently, it is desirable to replace the traditional stationary or well based thermal cyclers with continuous flow designs. A continuous flow polymerase chain reaction device consisting of a cylindrical heating core, which is segmented axially into three symmetric heating zones providing the denaturating, annealing and extension phases of the polymerase chain reaction, and a flow through capillary tube which is wound helically around the core has been fabricated and shown to consistently amplify target plasmid DNA samples. At the inlet to the device, PCR samples are segmented into droplets and entrained in an immiscible carrier fluid to eliminate cross contamination between PCR samples. This approach also prevents degradation of the micro-reactor droplets from inhibitory effects posed by the high surface to volume ratios associated with the device. The droplet train is then cycled through the capillary tube with each complete revolution constituting one cycle of the PCR reaction. The results reported in this paper include, initial validation of the spiral cycler design in comparison to an existing commercial PCR platform, and subsequent optimisation of the reaction time and its effects on the devices performance. The spiral thermal cycler has demonstrated successful PCR amplification at the nano scale with stable trains of 30-35nl droplet volumes being processed in an amplification time of 32 minutes. At this level the device offers the potential to process approximately 3500 such droplets in of order one hour.
UR - http://www.scopus.com/inward/record.url?scp=33846962510&partnerID=8YFLogxK
U2 - 10.1115/icnmm2006-96180
DO - 10.1115/icnmm2006-96180
M3 - Conference contribution
AN - SCOPUS:33846962510
SN - 0791847608
SN - 9780791847602
T3 - Proceedings of the 4th International Conference on Nanochannels, Microchannels and Minichannels, ICNMM2006
SP - 823
EP - 828
BT - Proceedings of the 4th International Conference on Nanochannels, Microchannels and Minichannels, ICNMM2006
PB - American Society of Mechanical Engineers
T2 - 4th International Conference on Nanochannels, Microchannels and Minichannels, ICNMM2006
Y2 - 19 June 2006 through 21 June 2006
ER -