Master of Science / Department of Animal Sciences and Industry / Dale A. Blasi / Effects of nucleotides (NA) (PSB Complex; DSS Global, Chicago, IL) with corn germ meal (CGM) or dried corn distillers grains (DDG) on growth performance, digestibility, in vitro ruminal gas production, and mucosal immunity were analyzed in 4 experiments. In Exp. 1, 213 crossbred heifers (BW= 262 ± 67.4 kg) were used in a complete block design with a 3 x 2 factorial arrangement of treatments to determine the net energy values of CGM in comparison to DDG and the effects of NA at three inclusion levels (0, 2, and 4 g) during an 84-d receiving period. Pens were randomly assigned to one of six treatments: 1) CGM with no NA (CGM0), 2) CGM with 2 g/heifer daily NA (CGM2), 3) CGM with 4 g/heifer daily NA (CGM4), 4) DDG with no NA (DDG0), 5) DDG with 2 g/heifer daily NA (DDG2), and 6) DDG with 4 g/heifer daily NA (DDG4). There were no significant effects of NA or the type of corn byproduct on growth performance (P ≥ 0.15). Exp. 2, was conducted to determine the performance and mucosal immunity effects of NA using 240 crossbred heifers (BW= 268 ± 34.1 kg). Pens were randomly assigned to three treatments which consisted of diets 4, 5 and 6 from Exp. 1. Calves were blocked by weight and assigned to a pen for 56-d. There were no significant effects of NA on growth performance results (P ≥ 0.18). On d 28, fecal samples were collected from approximately 5 calves from each pen and analyzed for secretory IgA concentration. NA inclusion did not affect fecal IgA concentration (P = 0.15). Exp. 3, utilized 4 ruminally cannulated Holstein heifers in a 4 x 4 Latin square design. The four treatments included diets 1 and 4 from Exp. 1 along with those two diets supplemented with 3 g/heifer daily NA. Ruminal pH increased as NA was included (P < 0.05). Ammonia concentrations were greater for DDG than for CGM (P < 0.01). Ruminal propionate concentration was less in diets that contained NA (P < 0.05). DDG diets led to greater concentrations of butyrate, isobutyrate, isovalerate, and valerate in ruminal fluid than CGM diets (P < 0.01). Valerate concentrations were decreased by NA when included in DDG diets, but not when added to CGM diets (interaction, P < 0.01). Isovalerate concentrations were increased by NA when included in CGM diets, but not when added to DDG diets (interaction, P = 0.01). An in vitro study, Exp. 4, evaluated 24-h gas production effects of the 6 treatments in Exp. 1. Gas production was decreased linearly by the inclusion of NA in DDG diets, but it was unaffected by NA in CGM diets (interaction, P < 0.01). CGM can be included in receiving and growing diets at 24.5% on a DM basis in place of DDG while maintaining growth performance, digestibility, and gas production. There was no effect of NA on growth performance, digestibility, or mucosal immunity, but there was an effect on ruminal gas production and ruminal parameters. Further research is needed to determine the effects of NA on receiving and growing cattle.

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.

As knowledge of the human genome has accelerated, various diseases and individuals’ responses to drugs have been pinpointed to specific DNA variations in one’s genome. Among many different types of variants, the most common and simplest is the single nucleotide polymorphism (SNP) in which a single base substitution occurs. Although there have been considerable improvements in technologies that can reveal a single base difference in a DNA strand, simple and affordable methods that have high detection sensitivity and require small sample volume are expected to facilitate widespread adoption of routine SNP analysis in clinical settings. One such method that meets these requirements is to use nanopore as a single molecule detector, an emerging analytic system that detects changes in current related to molecules occupying a nanometer aperture. This dissertation thus chronicles our endeavors in developing a nanopore-based SNP assay using polymer tagged dideoxynucleotides (ddNTPs). The fundamental principles of this method rely on single base extension (SBE) of a primer by DNA polymerase using polymer tagged ddNTP analogs for allele discrimination and simple electronic readout of an alpha hemolysin (αHL) nanopore current for allele detection at the single molecule level. Using four uniquely tagged ddNTPs, a characteristic current level that is specific to each base is produced, thus identifying the SNP alleles present and the genotype at the site. To demonstrate the feasibility of this approach, four polymer attached ddNTPs, each with a different tag that generates a characteristic current blockade level in the αHL nanopore, were designed and synthesized. To search for a DNA polymerase that can accept these tagged ddNTP analogs as substrates, several candidate DNA polymerases were surveyed and their relative efficiencies for incorporation of the analogs were compared (Chapter 2). To generate a steady and stable blockade event for accurate SNP analysis, two different means of positioning a tag molecule in the αHL nanopore after the SBE reaction have been explored: covalent conjugation of DNA primer to the pore and immobilization of biotinylated primer within the pore by streptavidin. To find a suitable position for primer attachment on the pore, three αHL mutants, each with a different single conjugation point, were constructed. Using these mutants, different DNA-pore conjugates were produced and purified via various chromatography systems (Chapter 3). In the nanopore system, charged molecules such as DNA are electrophoretically driven through the pore under an applied voltage, thereby modulating the ionic current through the nanopore. This current reveals useful information about the structure and dynamic motion of the molecule at the single molecule level. Before performing SNP analysis, we first studied single molecule behaviors of oligonucleotides of different lengths and structures in the αHL pore and their ensuing current signatures in the system (Chapter 4). Finally, harnessed with tools and insights from the nanopore single molecule studies, actual SNP assays were performed in our nanopore system using the polymer tagged ddNTPs and SBE. Chapter 5 discusses the integrated approach where SBE is achieved on a primer-conjugated αHL nanopore and Chapter 6 presents the results using a biotin-streptavidin complex for immobilization of tag molecules in the pore. Overall, this thesis validates adaptation of the nanopore detection system for SNP analysis using the polymer tagged ddNTPs.

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.

Thesis (Ph. D. in Chemical Engineering)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Patrick M. Gaffney, School of Marine and Earth Studies. Includes bibliographical references.

Graduation date: 1995

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.