Related Experiment Video
Updated: Aug 11, 2025

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
Published on: October 6, 2017
Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate
Shazeel Ahmad1, Syed Farhat Ali2, Saima Iftikhar1
1School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
Abstract:
Positively charged amino acids in the DNA polymerase domain are important for interaction with DNA. Two potential residues in the palm domain of Pca-Pol, a DNA polymerase from Pyrobaculum calidifontis, were identified and mutated to arginine in order to improve the properties of this enzyme. The mutant proteins were heterologously produced in Escherichia coli. Biochemical characterization revealed that there was no significant difference in pH, metal ion, buffer preferences, 3' - 5' exonuclease activity and error rate of the wild-type and the mutant enzymes. However, the specific activity, processivity and extension rate of the mutant enzymes increased significantly. Specific activity of one of the mutants (G522R-E555R) was nearly 9-fold higher than that of the wild-type enzyme. These properties make G522R-E555R mutant enzyme a potential candidate for commercial applications.
Related Concept Videos
Proofreading
Errors During Replication are Corrected by the DNA Polymerase...
PCR
Replication in Prokaryotes
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
Translesion DNA Polymerases
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
The Replisome
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
DNA Isolation

