Plastic versus glass capillaries for rapid-cycle PCR.
Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area-to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees -7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225-300 bp in length with melting temperatures of 90.3 degrees -93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.
Elenitoba-Johnson
Oluwole
O
University of Utah, Salt Lake City, Utah 84132, USA.
David
Derek
D
Crews
Niel
N
Wittwer
Carl T
CT
D001057:Apolipoproteins E / Q000235:genetics; D004867:Equipment Design; D004868:Equipment Failure; D015894:Genome, Human; D006358:Hot Temperature; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013814:Thermal Conductivity; D013997:Time Factors
D001057:Apolipoproteins E