A Fundamental Study of the PCR Amplification of GC-rich DNA Templates

Authors

T. G. Mamedov, University of Nebraska - Lincoln
Elsje Pienaar, University of Nebraska - LincolnFollow
Scott E. Whitney, University of Nebraska - LincolnFollow
Joel R. TerMaat, University of Nebraska-LincolnFollow
G. Carvill, University of Cape Town, South Africa
R. Goliath, University of Cape Town, South Africa
Anuradha Subramanian, University of Nebraska - LincolnFollow
Hendrik J. Viljoen, University of Nebraska - LincolnFollow

Date of this Version

12-2008

Comments

Published in Computational Biology and Chemistry 32:6 (December 2008), pp. 452-457; doi 10.1016/j.compbiolchem.2008.07.021 Copyright © 2008 Elsevier Ltd. Used by permission. http://www.elsevier.com/locate/compbiolchem

Abstract

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (η) of annealing as a function of temperature (T_A), annealing period (t_A), and template composition. The optimal efficiency lies in a narrow region of T_A and t_A for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX and HBB (with 78.72% and 52.99% GC, respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3–6 s and depend on annealing temperature. Annealing times greater than 10 s yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that shorter annealing times are not only sufficient but can actually aid in more efficient PCR amplification of GC-rich templates.