Date of Award:

5-1-2014

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biological and Irrigation Engineering

Advisor/Chair:

Anhong Zhou

Abstract

Cryptosporidium parvum is a deadly waterborne protozoan parasite that invades the gastrointestinal tract of humans and causes severe to life-threatening gastro enteric disease. Due to the ubiquitous nature of Cryptosporidium parvum in the world’s water, it is necessary to determine the source of an outbreak. Rapid detection and identification of various genotypes of Cryptosporidium are a valuable goal in determining the source of the pathogen in a human epidemic. Exploitation of gene sequences specific to species is a powerful tool detecting pathogens.

Molecular beacons are one of these tools for high selectivity and specificity detection of DNA and RNA. Molecular beacon is a single strand of DNA that forms a stem and loop structure, where the stem holds the DNA together and the loop detects a target sequence. This molecular beacon detection is determined by the changes of fluorescent emissions of fluorescent dye linked to the ends of the stem. In this thesis work, a new and novel molecular beacon was designed to detect the specific sequences from the heat shock protein gene of Cryptosporidium parvum that infects humans. This probe is synthesized by the conjugation of pyrene molecules to both ends of the stem which leads to a unique feature of pyrene excimer-monomer switching molecular beacon upon the hybridization of the loop sequence with the target DNA sequence.

This thesis systematically investigates the physical binding (e.g., quantum yield) and thermodynamic properties, including enthalpy, entropy, and free energy of this excimer-monomer switching molecular beacon in the presences of complimentary, mismatched, and damaged DNA, respectively, in the three phases: phase one is the molecular beacon in the stem and loop structure, phase two is the molecular beacon hybridized to its target DNA, and phase three is the molecular beacon in a random coil. The effect of magnesium concentration on the binding and thermodynamic properties was also investigated. Finally, as a comparison, a conventional fluorescence resonance energy transfer-based molecular beacon with a fluorophore at the 5’ end and quencher at the 3’ end was used to assess selectivity and sensitivity in detection of DNA-DNA hybridization.

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