INFRARED-MEDIATED THERMOCYCLING FOR ULTRAFAST POLYMERASE-CHAIN-REACTION AMPLIFICATION OF DNA

Interest in improving the speed of DNA analysis via capillary electrop horesis has led to efforts to integrate DNA amplification into microfa bricated devices. This has been difficult to achieve since the thermoc ycling required for effective polymerase chain reaction (PCR) is depen dent on an effective contact between the heating source and the PCR mi xture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chamb ers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected wit h a solenoid-gated compressed air source. With temperature ramping bet ween 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 t arget and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This s ets the stage for implementing DNA thermocycling into a variety of mic rofabricated formats for rapid PCR fragment identification and DNA seq uencing.