Figure 2.

Previously, Andrea Gochi prepared and analyzed three I614, E615G

double mutants: I614E, I614T, and I614M. Gochi found that the I614M double
mutant reduced the enzymes efficiency, while I614T didnt change it at all. The
I614E double mutant was more efficient than the lone E615G mutant. Image
adapted from Ref. 7.

A B
Figure 1. A) Physical model of Taq DNA polymerase. DNA is extended at the
active site (Yellow). B) Schematic displaying which positions on two bases,
pyrimidine and purine, can tolerate modifications. 2 modifications (Red) are
not tolerated by the wild type polymerase. Image adapted from Ref. 7.
References
Abstract
Characterization and comparison of mutant Taq DNA polymerase I
Nedim Filipovic, Hayley Schultz, Andrea Gochi, Aaron Leconte
Keck Science Dept of Claremont McKenna, Pitzer, and Scripps Colleges. Claremont, CA 91711
Introduction
Experimental Preparation Results
Conclusion
Acknowledgments
Aspartate (D) Lysine (K) Phenylalanine (F) Alanine (A)
Figure 3. A) Structure of Aspartate, displaying the amino acids characteristic
negative charge. B) Lysine. This amino acid was chosen for its positively
charged structure. C) Phenylalanine. Other directed evolution experiments
have created mutated Taq with the I614F mutation. It is also aromatic. D)
Alanine. This amino acid is the closest analog to an absence of an amino acid.
Image adapted from Ref 7.
A B C D
Figure 4. A diagram explaining the process of the polymerase chain reaction
(PCR). PCR can be used to introduce mutations into wild type DNA in a lab
environment. Image adapted from Ref. 6.
A B
Figure 6. A) After expressing cells containing the sought after gene, the cells
are lysed and the protein is separated. It is then flowed through a nickel
column, where the Histidine tag on Taq binds it to the nickel resin. A number of
buffer washes are used to purify the protein. B) The purification of the protein is
confirmed by an SDS page gel. The fractions (or washes) where the protein is
located are highlighted in red. Image adapted from Ref. 5.
Figure 5. A) The linear PCR product is then ligated to create a plasmid. This
form of ligation is called blunt-end ligation, referring to the blunt ends of the
linear DNA. B) The ligated DNA is then transformed into E. Coli cells, and
plated on an LB/Agar solution (antibiotic treated) to promote the growth of
colonies. Image adapted from Ref. 7.
B A
Figure 7. * indicates no enzyme was added (primer control). The farther the
coloring reaches up each column the more adenine bases the enzyme
incorporated during the reaction. It is clear to see that the I614E and I614D
double mutants were most efficient, proving more efficient than the E615G
single mutant. The other mutants were either similarly efficient or less efficient
than the single mutant.
The mutated enzymes were qualitatively assayed by Hayley Schultz. 1 nM
enzyme was incubated with DNA encoding six A additions and 2OH ATP for 60
minutes.
1. Ong, J.L., Loakes, D., Jaroslawski, S., Tpp, K. & Holliger, P. Directed evolution of
DNA polymerase, RNA polymerase and reverse transcriptase activity in a single
polypeptide. J Mol Biol 361, 537-50 (2006).
2. Fa, M., Radeghieri, A., Henry, A.A. & Romesberg, F.E. Expanding the substrate
repertoire of a DNA polymerase by directed evolution. J. Am. Chem. Soc. 126, 1748-
1754 (2004).
3. Xia, G. et al. Directed evolution of novel polymerase activities: Mutation of a DNA
polymerase into an efficient RNA polymerase. Proc. Natl. Acad. Sci. USA 99, 6597-
6602 (2002).
4. Hayley Schultz. Aaron M. Leconte (unpublished results).
5. Andrea Gochi. Aaron M. Leconte (unpublished results)
6. "Polymerase chain reaction" by Enzoklop - Own work. Licensed under Creative
Commons Attribution-Share Alike 3.0 via Wikimedia Commons
7. Aaron M. Leconte (unpublished work).

The transcription and replication of DNA is crucial to life. DNA polymerase
replicates DNA within an organism by extending bases along a template strand
of DNA. Polymerases have the ability to proofread nucleotides prior to
extension. 2 modifications can optimize nucleotides for therapeutic
applications. While wild type Taq DNA Polymerase does not tolerate
modifications to the nucleotides sugar in this position, mutated enzymes have
shown greater success. While previous experiments have shown that the
E615G mutation contributes most to the change in 2 modified nucleotide
incorporation efficiency. We aim to determine how the enzymes ability to
incorporate modified substrates responds to changes in the I614 amino acid.
This summer I focused on the effect of four additional I614X, E615G double
mutants: I614D, I614A, I614K, and I614F. Once the mutants were cloned and
prepared, they were qualitatively assayed. The reactions showed that the
I614A and I614K double mutants were less efficient at extending than the lone
E615G mutant. The I614F double mutant was similarly efficient to E615G.
Lastly, the I614D double mutant was more efficient than the single mutant, and
had similar data to the I614E double mutant.
Aaron Leconte, Sharon Chiang, Andrea Gochi, Constanza Jackson,
Jacqueline Kroll, Alexie Ogonowsky, Hayley Schultz, SCAMASS, and The Bill
& Melinda Gates Foundation.
It is clear that mutations at the 614 position in Taq DNA polymerase have an
affect on the enzymes ability to incorporate 2 modified nucleotides. The
results of my experiment show that, when paired with E615G, the I614E and
I614D mutants increased the enzymes efficiency greatly, making it more
efficient than the E615G single mutant. The I614F double mutant was similarly
efficient to the single mutant, while the I614A and I614K double mutants were
actually less efficient. This tells us that negatively charged amino acids at the
614 position have a positive effect on efficiency. Meanwhile, we can conclude
that positively charged and small amino acids actually decrease efficiency. This
information may be used to optimize Taq DNA polymerase for 2 modified
substrate incorporation. Future study of the impact that various single I614X
mutants have on Taqs efficiency are necessary to fully understand that
implications of these results. Furthermore, other amino acids need to be tested
to clarify some questions, such as the inactivity of phenylalanine.