Abigail Lopez's DNA Polymerase A12 Aptamer Project

A Polymerase Chain Reaction (PCR) cannot be run without the addition of some sort of “PCR Polymerase”. A commonly known and used polymerase is Taq DNA polymerase. Due to this enzyme being active at room temperature, it is common scientific practice to keep Taq and all other reagents on ice during prep/set up. Even when such precautions are taken nonspecific primer binding can occur. This ultimately leads to the generation of nonspecific products, which can significantly lower product yields in PCRs (Sigma-Aldrich, 2015). Despite PCRs being a process that most researchers have become quite familiar with, it seems as though poor amplification or PCR failure is a problem that many still encounter. A12 DNA polymerase and its possible aptamer could be the answer to that problem.

The target, A12 DNA polymerase, a dimer, has the molecular weight of 91.084kD. The target is depicted in Figure 1. It is a combination of Thermococcus kodakaraensis (KOD) and Pyrococcus furiosus (Pfu). The reasoning behind this combination was due to their high level of fidelity and distinct functional properties. Individually, KOD was more processive and faster, and Pfu had been shown to generate longer amplicons in routine PCR reactions, so it was thought that these properties combined would result in a very useful enzyme. It should be noted that the average error rate for Taq DNA Polymerase (and other Family A variants) is 1 in 103/104 nucleotides, which in comparison to the DNA Polymerases of Family B, such as A12, which averaged at 1 in 105 /106 nucleotides, are less accurate (Qiagen 2011). Taq continues to be used despite the increased chance of PCR failure and inaccuracy,

Discovering an aptamer for the target, A12 DNA Polymerase, which, at permissive temperatures, would dissociate from its inhibitor and commence polymerization, would significantly increase PCR accuracy by decreasing nonspecific priming and increasing product yields, this modified form of PCR is known as Hot Start PCR. One of the advantages of this modified PCR is that it helps avoid non-specific primer binding by inactivating the PCR polymerase at lower temperatures, a method of this, known as USB HotStart-IT, is depicted in Figure 2 (Sang et al., 2015).

Aptamers are nucleic acid species with high binding affinity for a specific target. Their discriminatory properties, such as being able to discriminate between related targets on the basis of a single amino acid or functional group, is just one of many advantages aptamers offer over antibodies. The application of an aptamer for this target, if discovered, would not be therapeutic or diagnostic, but would make an impact in the world of molecular biology, as it would be a new functional polymerase that would be available for use. The Ellington Lab, located at the University of Texas at Austin, is one of the labs also currently conducting aptamer research.

Kubu, Christopher J. (2008). HotStart-IT®: A Novel Hot Start PCR Method Based on Primer Sequestration. BioTechniques. Vol. 44 No. 2, 275

New England Biolabs Inc. DNA Polymerase Thermostability. (2015). DNA Polymerase Thermostability. New England Biolabs.

Qiagen. (2011). Maximizing PCR and RT-PCR success. Sample & Assay Technologies.

Sang, F., Yang, Y., Yuan, L., Ren, J., and Zhang, Z. (2015). Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots. Nanoscale 7, 15852-15862.

Sigma-Aldrich. (2015). Hot Start PCR. Sigma-Aldrich. Sigma-Aldrich Co. LLC.,