The kinetic requirements of extreme qPCR

Publication date: March 2019Source: Biomolecular Detection and Quantification, Volume 17Author(s): Adam L. Millington, Jessica A. Houskeeper, John F. Quackenbush, James M. Trauba, Carl T. WittwerAbstractThe kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq™), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom “extreme” PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200–500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10–60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short product...
Source: Biomolecular Detection and Quantification - Category: Molecular Biology Source Type: research