Gas jet process and apparatus for high-speed amplification of DNA
A gas, high-speed technology, applied in the field of gas injection polymerase chain reaction, which can solve the problem of lack of gas dynamics
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Embodiment 1
[0155] 30 PCR cycles in 5 minutes and 35 seconds (335 seconds)
[0156] Use pressurized air (214 bar = 36 p.s.i.) as "hot" gas and pressurized CO 2 (2.6 bar = 40 p.s.i.) was performed as a "cold" gas in a three-valve setup for gas jet DNA amplification experiments (Fig. 3a). Four model DNA templates of different lengths were PCR amplified in separate 10 μl reactions in thin-walled glass capillaries: (a) a 91bp E. coli O157:H7 Stx amplicon, (b) a 333bp λ"D "gene amplicon, (c) a 364bp human platelet antigen HPA-4 amplicon, and (d) a human β-globin 536bp amplicon, using pressurized air as hot gas and CO 2 Cool down and amplify using 30 cycles of [0 sec 92°C / 0 sec 55°C / 5 sec 72°C]. Gas-jet PCR reactions (10 μl) were performed in thin-walled glass capillary tubes containing 50 mM Tris (pH 8.5, 25°C), 250 μg / ml BSA, 3 mM MgCl 2 , 0.2 mM dNTPs, 50 pmol forward and reverse primers, 20 pg template DNA, and 5 U Taq polymerase (Promega, Madison, WI). After amplification, the reaction...
Embodiment 2
[0159] Software improvements (Refinements), 30 PCR cycles in 2 minutes 48 seconds (168 seconds)
[0160] The three-valve device is a functional thermal cycler. Not only is it very fast, but it also exhibits good thermal control (±1°C). However, the performance of the device is limited by its software written in BASIC code. Every time a command needs to be executed in response to a change in temperature in a reaction chamber or process heater, it needs to be translated from BASIC (interpreter) to assembly code (assembler).
[0161] Some time was wasted (~1 sec / loop) due to limitations of this software. In particular, more than 30 seconds of time (>1 second / cycle) were wasted unproductively during 30 cycles of gas-phase PCR amplification. Therefore, the system control software was rewritten in assembly code to run faster. This modification results in a faster gas-spray PCR.
[0162] At Taq polymerase extension rates ≥80 nt / s, it is expected that ~1 s / cycle spent during the ...
Embodiment 3
[0166] 30 PCR cycles in 1 minute 18 seconds (78 seconds)
[0167] A small (85bp) amplicon of the E. coli O157:H7 Stx gene was transferred using "hot" pressurized helium with CO 2 Cool for PCR amplification. To further reduce thermal cycling time, the primer length was increased to 30 nt so that higher annealing temperatures (62°C-63°C) could be applied. In addition, the DNA denaturation temperature was slightly lower (86°C-89°C) than that used in previous experiments.
[0168] Three different high-speed gas-phase thermal cycling protocols were applied: (a) [0 sec 89°C / 0 sec 62°C / 0 sec 72°C]; (b) [0 sec 87°C / 0 sec 62°C / 0 sec 72°C] and (c) [0 sec 86°C / 0 sec 63°C / 0 sec 72°C]. Perform PCR reaction (10 μl) in a thin-walled glass capillary tube containing 50 mM Tris (pH 8.5 25°C), 250 μg / ml BSA, 3 mM MgCl 2 , 0.2mMdNTPs, 50pmol forward and reverse primers, 20pg Escherichia coli O157:H7 DNA, and 5U of Taq polymerase (Promega, Madison, WI). At 30 times [0 seconds 89°C...
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