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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
A fundamental study of the PCR amplification of GC-rich DNA templates
Computational Biology and Chemistry, Volume 32, No. 6, Year 2008
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Description
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 (TA), annealing period (tA), and template composition. The optimal efficiency lies in a narrow region of TA and tA 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. © 2008 Elsevier Ltd. All rights reserved.
Authors & Co-Authors
Mamedov, Tarlan
United States, Lincoln
University of Nebraska–lincoln
Pienaar, Elsje
United States, Lincoln
University of Nebraska–lincoln
Whitney, Scott E.
United States, Lincoln
University of Nebraska–lincoln
TerMaat, Joel R.
United States, Lincoln
University of Nebraska–lincoln
Carvill, Gemma L.
South Africa, Cape Town
University of Cape Town
Goliath, R.
South Africa, Cape Town
University of Cape Town
Subramanian, Anuradha
United States, Lincoln
University of Nebraska–lincoln
Viljoen, Hendrik J.
United States, Lincoln
University of Nebraska–lincoln
United States, Omaha
985400 University
Statistics
Citations: 131
Authors: 8
Affiliations: 3
Identifiers
Doi:
10.1016/j.compbiolchem.2008.07.021
ISSN:
14769271
Research Areas
Genetics And Genomics
Study Approach
Quantitative