Researcher ORCID Identifier

0000-0001-8332-4219

Graduation Year

2021

Date of Submission

12-2021

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Molecular Biology

Reader 1

Aaron Leconte

Reader 2

Zhaohua Tang

Abstract

Chemically modified DNA (M-DNA) possesses several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize M-DNA, so in recent years M-DNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approach natural error rates and make M-DNA polymerases useful for applications that require high fidelity. In this project, mutation sets LVL and ETL, which have been shown to increase the fidelity of wild-type Taq polymerases, were added to some of the best current M-DNA polymerases (4-3, 4-6, and P1). The accuracies of these new mutants were then thoroughly characterized, and significant improvements of up to threefold were consistently measured relative to their parent enzymes. The resulting changes in polymerase activity resembled previous findings both in M-DNA and in wild-type Taq polymerases. These results will both improve M-DNA polymerase accuracy as well as inform future polymerase engineering efforts.

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This thesis is restricted to the Claremont Colleges current faculty, students, and staff.

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