High Speed Polymerase Chain Reaction in Constant Flow

Nakano, Hideo; Matsuda, Kōji; Yohda, Masafumi; Nagamune, Teruyuki; Endo, Isao; Yamané, Tsuneo

doi:10.1271/bbb.58.349

Cited by 75 publications

(47 citation statements)

References 2 publications

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“…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.…”

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Rapid PCR in a continuous flow device

et al. 2004

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Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 uC) and renaturation (55 uC-68 uC) zones; above 6 mm s 21 the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from l-DNA. Amplifications at velocities ranging from 1 mm s 21 to 20 mm s 21 were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle 21) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle 21 ). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).

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Rapid PCR in a continuous flow device

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“…It will also help chemical engineers to design an ideal PCR reactor. Nakano et al (1994) have fabricated and operated a continuous capillary reactor and have obtained an amplification yield of about half that obtained by a commercial thermocycler. However, they have not provided any mathematical treatment for the PCR process.…”

Section: Polymerase Chain Reaction (Pcr) Is An In Vitro Methodsmentioning

confidence: 99%

Polymerase chain reaction engineering

Hsu

Das

Mohapatra

1997

Biotechnol. Bioeng.

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“…The reader is also directed to related microminiaturization of PCR reactions in capillaries [9,10,11] and on the surface of microarrays [12,13,14].…”

Section: Introductionmentioning

confidence: 99%

Microchip PCR

Kricka

Wilding

2003

Analytical and Bioanalytical Chemistry

145

107

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Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties

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High Speed Polymerase Chain Reaction in Constant Flow

Cited by 75 publications

References 2 publications

Rapid PCR in a continuous flow device

Rapid PCR in a continuous flow device

Polymerase chain reaction engineering

Microchip PCR

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