Infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of DNA.
Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.
Oda
R P
RP
Department of Immunology, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Strausbauch
M A
MA
Huhmer
A F
AF
Borson
N
N
Jurrens
S R
SR
Craighead
J
J
Wettstein
P J
PJ
Eckloff
B
B
Kline
B
B
Landers
J P
JP
D004247:DNA / Q000737:chemistry*; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D019075:Electrophoresis, Capillary; D006358:Hot Temperature; D007259:Infrared Rays; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry*
D011948:Receptors, Antigen, T-Cell; D004247:DNA