Abstract
The 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 products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.
Highlights
As one of the simplest and most direct molecular tools available, Polymerase chain reaction (PCR) continues as a dominant force in molecular diagnostics
In addition to its extensive utility in research and diagnostics, qPCR is helpful in the study of PCR inhibitors and amplification efficiency [13]
Polymerase extension rates depend on many things, including the identity of the polymerase, buffer conditions, reactant concentrations, and temperature [14]
Summary
As one of the simplest and most direct molecular tools available, PCR continues as a dominant force in molecular diagnostics. Our understanding of how such a basic process works lags behind our enthusiasm to use it. The kinetic limits of PCR remain controversial and misunderstood. There is a mismatch between common instrumentation and the kinetic potential of PCR. Quantitative PCR and melting analysis can be performed in less than a minute [1], but most commercial protocols recommend over an hour. PCR speeds have increased over the years, most instruments today remain at slower, legacy speeds. Comparative speeds are best defined directly, either in the time for 1 cycle, or the total time for 30 cycles (Table 1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
