Rapid cycle DNA amplification: time and temperature optimization

Abstract

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.