Miniaturized flow-through PCR with different template types in a silicon chip thermocycler.
Flow-through chip thermocyclers can be used in miniaturized rapid polymerase chain reaction (PCR) despite their high surface to volume ratio of samples. We demonstrated that a thermocycler made of silicon and glass chips and containing thin film transducers for heating and temperature control can be adapted to the amplification of various DNA templates of different sources and properties. Therefore, the concept of serial flow in a liquid/liquid two-phase system was combined with a surface management of inner side walls of the microchannel and an adaptation of PCR mixture composition. In addition, the process temperatures and the flow rates were optimized. Thus, a synthetic template originating from investigations on nucleic acid evolution with 106 base pairs [cooperative amplification of templates by cross hybridization (CATCH)], a house keeping gene with 379 base pairs [glutaraldehyde 3-phosphate dehydrogenase (GAPDH)] and a zinc finger protein relevant in human pathogenesis with 700 base pairs [Myc-interacting zinc finger protein-1, knock-out (Miz1-KO)] were amplified successfully. In all three cases the selectivity of priming and amplification could be shown by gel electrophoresis. The typical amplification time was 1 min per temperature cycle. So, the typical residence time of a sample volume inside the 25 cycle device amounts to less then half an hour. The energy consumption of the PCR chip for a 35 min PCR process amounts to less than 0.012 kW h.
Schneegass
I
I
Institute for Physical High Technology (IPHT), Department of Biotechnical Micro Systems, Winzerlaer Str. 10, D-07745, Jena, Germany. schneegass@ipht-jena.de
Brautigam
R
R
Kohler
J M
JM
D001483:Base Sequence; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008904:Miniaturization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012825:Silicon / Q000737:chemistry; D013696:Temperature; D016335:Zinc Fingers
D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012825:Silicon