Title page for ETD etd-1104102-104609


Type of Document Dissertation
Author Farquar, Hannah D.
URN etd-1104102-104609
Title The Ligase Detection Reaction: The Evolution of a Mutation Detection Strategy
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Robert P. Hammer Committee Chair
Frank Cartledge Committee Member
Paul Russo Committee Member
William Crowe Committee Member
Thomas Gillis Dean's Representative
Keywords
  • dna polymerase
  • ldr
  • nucleoside analog
  • dna
Date of Defense 2002-10-23
Availability unrestricted
Abstract
Early detection of genetic mutations is important for control of diseases such as cancer and Alzheimer's. Early detection requires methods that detect small amounts of mutated DNA in very large amounts of normal or wild type DNA. One method to detect mutated DNA is the ligase detection reaction (LDR). Since its inception LDR has evolved greatly from a simple detection reaction after PCR amplification to PCR/RE/LDR, a scheme which uses nucleoside base analogs in PCR to convert wild type sequences to sequences containing restriction endonuclease (RE) sites which can then be cleaved leaving only mutant sequences for detection by LDR. Analysis of LDR has also evolved from slab gel electrophoresis to microarray analysis.

Understanding the structure and DNA polymerase recognition of nucleoside base analogs used in PCR/RE/LDR is key to improving this detection scheme. The use of higher fidelity DNA polymerase containing 3'→5' exonuclease domains for error correction is also important in early detection of genetic diseases. Pyrazole-based nucleoside analogs have been studied computationally and enzymatically. The stability a DNA containing these analogs depends largely on the dipole moment of the analogs, rather than polarizability or surface area. Reduced DNA polymerase recognition is due in part to altered base pair geometry, either inherent or created by DNA polymerase. Thiazole and thiazole N-oxide analogs to be used in the PCR/RE/LDR assay have been synthesized and characterized computationally, thermodynamically, and enzymatically. The N-oxide, a pyrimidine O2 mimic, enhances DNA stability and DNA polymerase recognition. The N-oxide increases electrostatic properties and solvation by the formation of a hydrogen bond when base paired with guanine. Enzymatic analysis indicated a preference for the base pairing of thiazole N-oxide with guanine and thiazole with adenine. An N3'→P5' phosphoramidate backbone analog has shown to inhibit the exonuclease activity of higher fidelity DNA polymerases for use in PCR/RE/LDR.

The evolution of the analysis of LDR continues with the adaptation to capillary and microdevice electrophoresis. These formats were used to analyze model samples and LDR reactions mimicking low abundant mutations. These improved techniques greatly improve the resolution of LDR analysis.

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