Karina Gomez's DNA Polymerase (A12) Aptamer Project

Replication performed by DNA polymerases at unwanted times and locations on the template, due to the temperature conditions that it is exposed to, can become a huge issue for researchers. The time required for reactions becomes even more extended as errors, after PCR reactions, become present. As a researcher, one can support the fact that one of the main reasons for malfunctioning PCR reactions is due to the Taq DNA polymerases’ tendency to start replicating when it reaches a certain temperature (slightly lower than room temperature) at an unwanted time. Not only does it begin replicating when it is exposed to room temperature, but sometimes even when it is placed on ice. One has to work quickly by retrieving the DNA polymerase from the -20 degree Celsius fridge, instantly add the correct amount into one’s reactions, mix the DNA polymerase along with the other reagents already present, and perform PCR as soon as it is added while trying to not create a malfunctioning reaction. These extra steps must be taken in order to reassure the reaction has not started replicating until PCR has initiated in the thermocycler, which can become quite troublesome.

In order to prevent failure in reactions, an antibody-like structure, must be used to halt the DNA polymerase from replicating. In this case, instead of using antibodies, which are less reliable, more expensive, and difficult to perform with, an Aptamer will be used in its place. Aptamers are nucleic acid binding species that selectively bind to molecules, in this case proteins to perform wanted functions. Aptamers bind to specific targets with high specificity through its nitrogen base sequences which fold into complex shapes to perform interactions and stay tightly bounded with the targets. Aptamers are used for diagnostics in locating diseases by binding to the protein and preventing it from predominating. They can also be used in therapeutics such as binding to a protein and preventing it from taking over and further damage.

Aptamer selection is essential for conducting these important procedures. RNA and DNA pools behave in a similar pattern as how nature performs Natural Selection. As one performs more and more rounds of selection, the tightest/ strongest RNA sequences that bind to the target are found after undergoing multiple washes.

The purpose of this experiment is to find an aptamer this experiment, instead of using Taq polymerase which is derived from the E.Coli bacteria, the DNA polymerase that was used was the A12 DNA polymerase was derived in a laboratory by biomechanically joining two DNA polymerases called Thermococcus kodakareensis and Pyracoccus furiosus. DNA Polymerases B,C and D are the three main types of DNA polymerases which all have different characteristics. In this study, a biomechanical form of Polymerase B will be used to find an aptamer against it. DNA Polymerase B is responsible in replicating the chromosomal DNA as well as in the genome in the Archaea domain in the eukaryotic domain. A study was done at the University of Texas on two family B DNA polymerases called Thermococcus kodakareensis, and Pyracoccus furiosus These two DNA polymerases were combined together to create what is known as the A12 DNA Polymerase from the B family Archaea, which is what this experiment will be working with. This target is heteroduplex because it is a double stranded molecule that is from two different organisms. The two Archaea organisms’ DNA polymerases were combined together due to the fact that KOD is faster, and Pfu has shown to generate longer amplicons in routine PCR reactions, (Article: Directed Evolution of Archaeal Family B DNA Polymerase Improves Function in Bulk and Emulsion PCR). Therefore, the A12 DNA Polymerase was biomechanically made to be bound to a DNA template.

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