Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Karl E. Wommack, Dept. of Plant & Soil Sciences. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (B.S.) Summa Cum Laude--Butler University, 2007. / Includes bibliographical references (leaves 30-31).

A 1.57 kb XhoI restriction fragment derived from the TOL plasmid pDKI was subcloned into the E. Coli plasmid pUC19. The complete nucleotide sequence of this XhoI fragment was determined using both the chemical cleavage and chain termination DNA sequencing methods.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Computer Science / Doctoral / Doctor of Philosophy

by Agnes Pui-Yee Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 162-177). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.vii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Physiology of growth --- p.3 / Chapter 1.2 --- The anterior pituitary --- p.4 / Chapter 1.3 --- Chemistry of GH and the GH gene family --- p.7 / Chapter 1.4 --- Biochemical effects and mode of action of GH --- p.8 / Chapter 1.5 --- Control of GH at cellular level --- p.10 / Chapter 1.6 --- Control of GH gene expression at molecular level / Chapter 1.6.1 --- Introduction --- p.11 / Chapter 1.6.2 --- Tissue-specific expression of GH gene / Chapter 1.6.2.1 --- Tissue-specific transcription factors of pituitary cells --- p.20 / Chapter 1.6.2.2 --- Non-tissue specific transcription factors of pituitary cells --- p.27 / Chapter 1.6.2.3 --- Negatively-acting transcription factors of non-pituitary cells --- p.34 / Chapter 1.6.2.4 --- Theory for tissue-specific GH gene activation --- p.39 / Chapter 1.7 --- Characteristic of growth in fish --- p.40 / Chapter 1.8 --- Objectives of the present study --- p.42 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Preparation of DNA / Chapter 2.1.1.1 --- Minipreparation of DNA --- p.46 / Chapter 2.1.1.2 --- Preparation of DNA using Qiagen column --- p.47 / Chapter 2.1.1.3 --- Preparation of phage DNA --- p.48 / Chapter 2.1.2 --- Elution of DNA from agarose gel --- p.51 / Chapter 2.1.3 --- Preparation of competence cells and transformation --- p.52 / Chapter 2.1.4 --- Ligation of DNA fragments --- p.53 / Chapter 2.1.5 --- Cell feeding and subculturing --- p.54 / Chapter 2.2 --- Special techniques / Chapter 2.2.1 --- DNA sequencing --- p.56 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.67 / Chapter 2.2.3 --- Direct sequencing of PCR products --- p.72 / Chapter 2.2.4 --- Nested-deletion --- p.75 / Chapter 2.2.5 --- DNA transfection --- p.81 / Chapter 2.2.6 --- CAT assay --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Sequencing of the grass carp GH gene / Chapter 3.1.1 --- Introduction --- p.93 / Chapter 3.1.2 --- Sequencing strategy --- p.94 / Chapter 3.2 --- Sequence analysis of the grass carp GH gene --- p.108 / Chapter 3.3 --- Functional analysis of the grass carp GH gene --- p.115 / Chapter CHAPTER 4 --- DISCUSSIONS / Chapter 4.1 --- DNA sequence comparison between grass carp GH gene and other organisms --- p.137 / Chapter 4.2 --- Amino acid comparisons between grass carp GH and other organisms --- p.143 / Chapter 4.3 --- Tissue-specific expression of GH gene / Activation of transcription --- p.154 / Repression of transcription --- p.155 / Chapter 4.4 --- Electroporation of zebrafish eggs --- p.157 / Chapter 4.5 --- Further studies --- p.160 / REFERENCES --- p.162 / APPENDIX --- p.178

Thesis (M.Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2002. / Includes bibliographical references (p. 83-86). / DNA sequencing is the process of determining the sequence of chemical bases in a particular DNA molecule-nature's blueprint of how life works. The advancement of biological science in has created a vast demand for sequencing methods, which needs to be addressed by automated equipment. This thesis tries to address one part of that process, known as base calling: it is the conversion of the electrical signal-the electropherogram--collected by the sequencing equipment to a sequence of letters drawn from ( A,TC,G ) that corresponds to the sequence in the molecule sequenced. This work formulates the problem as a pattern recognition problem, and observes its striking resemblance to the speech recognition problem. We, therefore, propose combining Hidden Markov Models and Artificial Neural Networks to solve it. In the formulation we derive an algorithm for training both models together. Furthermore, we devise a method to create very accurate training data, requiring minimal hand-labeling. We compare our method with the de facto standard, PHRED, and produce comparable results. Finally, we propose alternative HMM topologies that have the potential to significantly improve the performance of the method. / by Petros T. Boufounos. / M.Eng.and S.B.

High-throughput DNA sequencing technology has advanced rapidly in the past few decades and is the driving force for personalized precision medicine. In this Thesis, a set of novel disulfide linker-based nucleotide reversible terminators (NRTs) has been designed and synthesized for application in DNA sequencing by synthesis (SBS), which is the dominant sequencing platform. The design and synthesis principles are outlined as follows. Four nucleotides (A, C, G, T) are modified as NRTs for the DNA extension reaction catalyzed by polymerase by attaching a cleavable fluorophore to a specific location on the base and blocking the 3′-OH group with a small chemically-reversible moiety so that the resulting molecules are still recognized by DNA polymerase as substrates. In these fluorescent NRTs, the fluorophores are attached through a disulfide (-SS-) cleavable linker to the 5-position of cytosine and thymine, and to the 7-position of deaza-adenine and deaza-guanine, and a small disulfide moiety is used to cap the 3'-OH group of the deoxyribose. The resulting fluorescent NRTs (3′-O-tert-butyldithiomethyl-dNTP-SS-fluorophores) are shown to be good substrates in DNA polymerase catalyzed reactions. The fluorophore and the 3′-O-tert-butyldithiomethyl group on a DNA extension product, which is generated by incorporating the 3′-O-tert-butyldithiomethyl-dNTP-SS-fluorophore in a polymerase reaction, are removed simultaneously and rapidly by treatment with a reducing agent, tris (3-hydroxypropyl) phosphine, in aqueous buffer solution. This one-step dual-cleavage reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner. Compared with existing fluorescent NRTs, the unique disulfide linkers used to synthesize the NRTs described in this thesis are cleaved efficiently under DNA compatible conditions, leading to shorter scars on the DNA extension strand to further improvement of the DNA SBS technology.

This dissertation describes the design and synthesis of novel cleavable fluorescent/anchor modified nucleotide reversible terminators using 3’-O-dithiomethyl (3’-O-DTM; 3’-O-SS) as a linker to directly or indirectly attach a fluorescent reporter to achieve scarless DNA Sequencing by Synthesis (SBS). To develop these nucleotide analogues for four-color SBS, two nucleotide analogues (3’-O-ROX-SS-dATP and 3’-O-BodipyFL-SS-dTTP) with directly attached fluorescent dyes and two other nucleotide analogues with directly attached biotin or trans-cyclooctene (TCO) as anchors (3’-O-Biotin-SS-dCTP and 3’-O-TCO-SS-dGTP) were successfully designed and synthesized. The nucleotide analogues with a PEG-elongated linker (3’-O-ROX-PEG4-SS-dATP, 3’-O-BodipyFL-PEG4-SS-dTTP, 3’-O-Biotin-PEG4-SS-dCTP and 3’-O-TCO-PEG4-SS-dGTP) were also designed and synthesized to optimize their incorporation efficiency in polymerase reactions. In our design, Biotin and TCO were demonstrated to be anchor moieties with high efficiency and specificity for binding with fluorescently labeled streptavidin and tetrazine, respectively. The DNA extension products produced by polymerase incorporation of 3’-O-Biotin-SS-dCTP and 3’-O-Biotin-PEG4-SS-dCTP were accurately identified by binding to Cy5-labeled streptavidin, while the DNA extension products produced by polymerase incorporation of 3’-O-TCO-SS-dGTP and 3’-O-TCO-PEG4-SS-dGTP were identified with equal precision by reaction with TAMRA-labeled tetrazine. A proof-of-concept experiment was conducted to demonstrate four-color scarless SBS using the novel nucleotide analogues described above.