Marie Diaz's DNA Polymerase (A12) Aptamer Project

Hot-Start to PCR Amplification: An Aptamer Selection against “A12” DNA Polymerase

Introduction & Background

DNA polymerase is the primary enzyme used in polymerase chain reactions (PCR) to produce an identical copy of the existing “template” strand of DNA, and it is most often derived from Thermus aquaticus (Taq). PCR is used to amplify specific sequences of DNA at one time, and is able to yield millions of copies per reaction. Approximately 2n copies of a particular sequence can be produced, where n represents the number of PCR cycles performed. Since its development in the late 1980’s by Kary Mullis, the PCR technique has allowed for advancements in the realm of genetic testing and even the detection of the AIDS virus, leading Mullis to win a Nobel Prize in 19931. The reaction includes primers, amongst many other reagents, which are fragments of single-stranded DNA that the polymerase utilizes to create a complementary strand of DNA (Figure 1). 3

However, there are many drawbacks to conventional polymerase chain reactions. Specifically, the catalysis of non-specific priming is due to the time between the original combination of reactants and the temperature elevation of the reaction necessary for the denaturing process, which allows the PCR to begin. Since the DNA polymerase in the human body would be unable to withstand such temperatures required in a laboratory setting, DNA polymerase is derived from thermostable bacteria, such as Thermus aquaticus. However, it still functions significantly at about 20-37°C. This can cause “primer dimer” formation, in which primers serve as templates for one another, causing them to bind together. These unwanted forms of DNA synthesis can lead up to a 50x decrease in accuracy and efficiency, especially for PCR that require a decent amount of cycles to properly amplify.4 Although DNA polymerase derived from Thermus aquaticus remains widely used, other thermophilic bacterial sources have shown higher levels of fidelity within in PCR. These sources include Thermococcus kodakaraensis (KOD) and Pyrococcus furiosus (Pfu) (Figure 2). 5 Unlike Taq, both KOD and Pfu have active 3’-5’ exonuclease activity, classifying them as proofreaders.4

These Family B polymerases- KOD DNA polymerase and Pfu DNA polymerase are able to process replication in a shorter amount of time, and hold larger elongation rates, respectively. The Ellington Lab at The University of Texas at Austin created “A12” DNA polymerase (Figure 3), the product of DNAP shuffling of KOD and Pfu DNA polymerases through recombination.6

In order to eliminate unnecessary DNA synthesis in PCR, the activity of DNA polymerase must be inhibited below the specific annealing temperatures. This would result in the creation of true “Hot-Start” PCR mechanism, in which the polymerase would only activate at the annealing temperature or higher temperatures. Recent aptamer research at The University of Texas at Austin appears promising.

“Aptamers” are nucleic acids with outstanding binding affinity for certain targets which include proteins, polypeptides, various ions, and other small molecules, which makes them more efficient than antibodies. Since they are cost-effective and regularly synthesized, they have been used in a wide variety of areas, including the development of innovative methods for drug delivery, therapeutics, and diagnostics. There are many methods of aptamer selection, including bead panning, filtration, and chromatography. Bead based selection is one method of in vitro selection in which the pool of potential aptamers is enriched through rounds of selection and amplification.

Through Nickel bead-based selection, PCR, gel electrophoresis and transcription, the discovery of an aptamer against “A12” DNA polymerase is expected. This aptamer would ideally inhibit “A12” DNA polymerase capabilities below an optimally selected annealing temperature of 57°C in this experiment. Once that temperature was reached, the aptamer against “A12” would then “fall off” and the PCR would start from that point rather than lower temperatures. Since the “A12” polymerase has a molecular weight of 91.084 kDa and an Isoelectric Point (PI) of about 8.37, it is negatively charged within its selection buffer with a pH of 8.8. Since DNA is negative, it will be less likely for the polymerase to bind to the DNA. A specialized ‘A12’ selection buffer is utilized so that the ideal aptamer binds to the polymerase through ’negative selection.’ Since “A12” is the byproduct of KOD and Pfu, it is important to note that both of those molecules are thermostable and therefore are able to resist higher temperatures. A12 is his-tagged so that it may bind to nickel beads used in the selection process, rather than wash away with the nonspecific proteins. The six polyhistadine tag (his-tag) is “easily incorporated” to the C or N terminus through “site-directed mutagenesis or PCR methods.

Attempts to create an aptamer that results in the development of a “Hot-Start” PCR have failed. However, this does not suggest that a “Hot-Start” method will fail to be developed through the discovery of an aptamer against A12. This Hot-Start technique could improve many fields involved with PCR reactions, including cancer detection, genetic testing, paternity testing, oral disease mechanisms, and crime scene investigations.8 The focus has rested on PK6 DNA polymerase, which is similar to A12 DNA polymerase because it was also created from KOD and Pfu in the Ellington Lab.

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References

[1] "Roche and PCR: A Monumental Scientific Discovery." History of PCR. Roche Molecular Systems. 13 Sept. 2015

[2] Carr, Steven M. "The Polymerase Chain Reaction." PCR. 2014. 14 Sept. 2015.

[3] "The History of PCR." Smithsonian Institution Archives. Smithsonian Videohistory Collection, 25 Feb. 1993. 9 Apr. 2015.

[4] McInerney, Peter, Paul Adams, and Masood Z. Hadi. "Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase." Molecular Biology International (2014): 1-8. Web.

[5] Cline, J. "PCR Fidelity of Pfu DNA Polymerase and Other Thermostable DNA Polymerases." Nucleic Acids Research 24.18 (1996): 3546-551.

[6] Ellington, Andrew, and Gwendolyn Stovall. Directed Evolution of Archaeal Family B DNA Polymerases Improves Function in Bulk and Emulsion PCR. Austin: Ellington Lab, 2015. Print.

[7] Bornhorst, Joshua A., and Joseph J. Falke. "[16] Purification of Proteins Using Polyhistidine Affinity Tags." Methods in Enzymology Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification 326 (2000): 245-54. PubMed. Web. 28 Sept. 2015.

[8] Benson, Linda. "Advantages of Thermococcus Kodakaraensis (KOD) DNA Polymerase for PCR-Mass Spectrometry Based Analyses."American Society for Mass Spectrometry 14 (2003): 601-04.

[9] Goff, Lindsey K., Michael J. Neat, Charles R. Crawley, Louise Jones, Emma Jones, T. Andrew Lister, and Rajnish K. Gupta. "The Use of Real-time Quantitative Polymerase Chain Reaction and Comparative Genomic Hybridization to Identify Amplification of the REL Gene in Follicular Lymphoma." British Journal of Hematology 111.2 (2000): 618-25

[10] Hashimoto, Hiroshi. "Crystal Structure of DNA Polymerase from Hyperthermophilic Archaeon Pyrococcus Kodakaraensis KOD1 1."Journal of Molecular Biology 306.3 (2001): 469-77.

[11] "High Dynamic Range and High Sensitivity QPCR and QRT-PCR." Gene. Gene Company Ltd. 12 Apr. 2015.

[12] "PCR." PCR: Learn Genetics. University of Utah Health Sciences, 2015. 14 Sept. 2015

[13] Kermekchiev, M. B. "Cold-sensitive Mutants of Taq DNA Polymerase Provide a Hot Start for PCR." Nucleic Acids Research 31.21 (2003): 6139-147. Oxford Journals. 11 Apr. 2015.

[14] "Toyobo Life Science Department." High Fidelity & Efficient PCR Enzyme: KOD DNA Polymerase. 12 Apr. 2015.

[15] "Reagents for Functional Genomics." National Center for Biotechnology Information. U.S. National Library of Medicine. 11 Apr. 2015.