Browsing by Subject "DNA microarrays"
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Item Computational and experimental methods in functional genomics : the good, the bad, and the ugly of systems biology(2008-08) Hart, Glen Traver; Marcotte, Edward M.Seven years into the postgenomic era, we sit atop a mountain of data whose generation was enabled by gene sequencing. The creation, integration, and analysis of these large scale data sets allow us to move forward toward the complementary goals of determining the individual roles of the thousands of uncharacterized mammalian genes and understanding how they work together to produce a healthy human being -- or, perhaps more importantly, how their malfunction results in disease. Collapsing the results of large-scale assays into gene networks provides a useful framework from which we can glean information that advances both of these goals. However, the utility of networks is limited by the quality of the data that goes into them. This study offers seeks to shed some light on the quality and breadth of protein interaction networks, describes a new experimental technique for functional genetic assays in mammalian cell lines, and ultimately suggests a strategy for how to improve the overall utility of the output generated by the systems biology community.Item The development of a microbead array for the detection and amplification of nucleic acids(2006) Ali, Mehnaaz Fatima; McDevitt, John T.The focus of this doctoral thesis is on the development of a chip-based sensor array, composed of individually addressable agarose micro-beads, that is suitable for the real-time detection of DNA oligonucleotides. This research is consistent with recent trends in disease diagnostics following the miniaturization and integration of sample preparation and measurement steps towards portable devices capable of point of care analysis. Thus, the power and utility of this microbead array methodology for DNA detection is demonstrated here for the analysis of fluids containing a variety of similar short oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10,000 and limit of detection values of ~10-13 M are obtained readily with this microbead array system. These analytical characteristics, here exhibited are competitive with some of the best direct DNA detection methodologies before reported. As an extension of this work, an integrated self quenching based sensing system within the bead format has shown clear efficacy for the detection of HIV gag isolates and Bacillus anthracis (Sterne) purified strains and allows for the rapid detection of 100bp sequences with sensitivities in the subnanomolar range. Additionally, due to the tailored immobilization of specific sequences on each sensor element, the multiplexed detection of various sequences utilizing diverse strategies has been demonstrated. Use of the micro-bead array in tandem with the hybridization capabilities of molecular beacons, constitutes a powerful tool for the heterogeneous elucidation of specific sequences. Concomitantly, successful collaboration with the Chen group on the development of a miniaturized enzyme based nucleic acid amplification device has been reported. Purified strains of Bacillus anthracis (Sterne) have been successfully amplified by the miniaturized polymerase chain reaction (PCR) chip as seen by gel electrophoresis. One of the long term aims of this general area of research will be to couple the glass micro chip-based PCR amplification of oligonucleotides with the real-time detection capabilities of a bead based array. These efforts serve to establish some precedent for the bead-based microfluidics approach to be implemented in the context of genomics testing for the next generation of health care.Item Development of imaging-based high-throughput genetic assays and genomic evaluation of yeast gene function in cell cycle progression(2007-12) Niu, Wei; Marcotte, Edward M.Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.Item Development of microdevices for applications to bioanalysis(2007) Kim, Joohoon, 1976-; Crooks, Richard M. (Richard McConnell)The development of microdevices for applications related to bioanalysis is described. There are two types of microdevices involved in this study: DNA (or RNA) microarrays and bead-based microfluidic devices. First, a new method to fabricate DNA microarrays is developed: replication of DNA microarrays. It was shown that oligonucleotides immobilized on a glass master can hybridize with their biotin-modified complements, and then the complements can be transferred to a streptavidinfunctionalized replica surface. This results in replication of the master DNA array. Several innovative aspects of replication are discussed. First, the zip code approach allows fabrication of replica DNA arrays having any configuration using a single, universal master array. It is demonstrated that this approach can be used to replicate master arrays having three different sequences (spot feature sizes as small as 100 [mu]m) and that master arrays can be used to prepare multiple replicas. Second, it is shown that a surface T4 DNA polymerase reaction improves the DNA microarray replication method by removing the requirement for using presynthesizd oligonucleotides. This in-situ, enzymatic synthesis approach is used to replicate DNA master arrays consisting of 2304 spots and arrays consisting of different oligonucleotide sequences. Importantly, multiple replica arrays prepared from a single master show consistent functionality to hybridization-based application. It is also shown that RNA microarrays can be fabricated utilizing a surface T4 DNA ligase reaction, which eliminates the requirement of modified RNA in conventional fabrication schemes. This aspect of the work shows that the replication approach may be broadly applicable to bioarray technologies. A different but related aspect of this project focuses on biosensors consisting of microfluidic devices packed with microbeads conjugated to DNA capture probes. The focus here is on understanding the parameters affecting the hybridization of DNA onto the probeconjugated microbeads under microfluidic flow conditions. These parameters include the surface concentration of the probe, the flow rate of the solution, and the concentration of the target. The simple microfluidic device packed with probe-conjugated microbeads exhibits efficient target capture resulting from the inherently high surface-area-to-volume ratio of the beads, optimized capture-probe surface density, and good mass-transfer characteristics. Furthermore, the bead-based microchip is integrated with a hydrogel preconcentrator enhancing the local concentration of DNA in a icrochannel. The integration of the preconcentrator into the bead-based capture chip allows significantly lower limit of detection level (~10-fold enhancement in the sensitivity of the microbeadbased DNA detection).Item Higher-order generalized singular value decomposition : comparative mathematical framework with applications to genomic signal processing(2010-08) Ponnapalli, Sri Priya; Ghosh, Joydeep; Alter, Orly, 1964-; Beckner, William; Caramanis, Constantine; Evans, Brian L.; Van Loan, CharlesThe number of high-dimensional datasets recording multiple aspects of a single phenomenon is ever increasing in many areas of science. This is accompanied by a fundamental need for mathematical frameworks that can compare data tabulated as multiple large-scale matrices of di erent numbers of rows. The only such framework to date, the generalized singular value decomposition (GSVD), is limited to two matrices. This thesis addresses this limitation and de fines a higher-order GSVD (HO GSVD) of N > 2 datasets, that provides a mathematical framework that can compare multiple high-dimensional datasets tabulated as large-scale matrices of different numbers of rows.Item RNA aptamer microarrays for the specific detection of proteins and their potential use as molecular diagnostics for the treatment of HIV(2006) Collett, James Raymond; Ellington, Andrew D.Aptamers are nucleic acid species with specific binding properties that are selected in vitro from large pools of random oligonucleotides. In the present work, I adapted microarray technology for the production of DNA oligonucleotide microarrays to print biotinylated RNA aptamer clones and pools onto streptavidin coated microarray slides in ways that preserved the specific protein binding functionalities of the aptamers. The resulting RNA aptamer microarrays could reliably detect fluorescently labeled HIV- 1 reverse transcriptase (RT), hen egg white lysozyme, and other proteins in a dosedependent manner, with linear signal responses that spanned up to 7 orders of magnitude of analyte concentration, and lower limits of detection in the pg/ml range. Aptamers on the microarray retained their specificity for target proteins in the presence of a 10,000 fold (w/w) excess of T-4 cell lysate protein. Aptamer microarray development was facilitated by the optimization of high-throughput and highly parallel methods for the enzymatic synthesis of 5’ biotinylated RNA aptamer clones and pools. As an alternative, unmodified RNA aptamers were prepared for immobilization by appending an 18 nt generic linker sequence to their 3’ ends that was hybridized to a 5’ biotinylated LNA/DNA anchor oligonucleotide, thereby forming a duplex with a Tm of 80o C. Applications for RNA aptamer microarrays as molecular diagnostics for HIV-1 treatment were potentiated by detecting the specific capture of unlabeled RT on the microarrays using fluorescent antibody sandwich methods. Microarray tests of wild type and drugresistant RT produced recombinantly in our laboratory indicated a nascent ability for RNA aptamers to distinguish between closely related RT phenotypes, thereby further demonstrating the potential utility of RNA aptamer-based diagnostics in the treatment of HIV.Item RNA profiling in an Alzheimer's disease mouse model(2008-08) Bao, Hongbo, 1977-; Harris, R. Adron; Mayfield, R. DayneAlzheimer’s disease (AD) is one of the common diseases of older people. Although several genes have been identified for Familial Alzheimer’s Disease (FAD), a reliable diagnostic, especially for those patients in their early or intermediate phases of AD, is still not available. There is neither effective treatment nor drugs that can stop or reverse AD progression. Breakthroughs in diagnosis or treatment development likely require understanding of the molecular mechanisms of AD. Studies in FAD have shown that APP, PS1, PS2 and some other genes are related to FAD. Mutations of APP and PS1 lead to amyloid plaque accumulation which is also prominent in Sporadic AD. Transgenic animals closely mimic human AD pathologies in many aspects. A mouse model carrying both APP Swedish mutation and PS1 deltaE9 mutation is used in this study. This mouse model accumulates amyloid plaque rapidly, and the plaque shows up as early as 6 months of age. Using microarrays, we have isolated 176 genes with significant expression changes and 14 turned on/off genes from AD mouse cortex. From this cDNA microarray measurement of global gene expression, several functional groups were regulated significantly in our mouse model of AD pathology. Mt2 and Atp7a were identified and may be candidates for further studies of AD pathology, as well as potential drug targets. Five significant microRNAs were found from AD mouse cortex, providing evidence that microRNAs could play a role in AD. cDNA arrays were also used to identify potential biomarkers from whole blood samples that distinguish AD mice from their non-transgenic littermates.Item Tensor generalizations of the singular value decomposition for integrative analysis of large-scale molecular biological data(2007-12) Omberg, Larsson Gustaf, 1977-; Alter, Orly, 1964-; Sitz, Greg OrmanThe structure of large-scale molecular biological data is often of an order higher than that of a matrix, especially when integrating data from different studies. Flattened into a matrix format, much of the information in the data is lost. I describe the use of higher-order generalizations of singular value decomposition (SVD) - both the higher-order singular value decomposition (HOSVD) and Parallel Factorization (PARAFAC) - in transforming tensors into simplified spaces. I apply these transformations to a series of DNA microarray datasets from different studies tabulated in a tensor of genes × time × conditions, specifically an integration of genome-scale mRNA expression data from three yeast-cell cycle time courses. One of the time courses was under exposure to the oxidative stress agent hydrogen peroxide (HP); another was exposed to menadione (MD) and the third was unstressed[45]. The HOSVD transforms the tensor to a “core tensor” of “eigenarrays” × “timeeigengenes” × “condition-eigengenes,” where the eigenarrays, time-eigengenes and condition-eigengenes are unique orthonormal superpositions of the genes, times and conditions, respectively. This HOSVD, also known as N-mode SVD, formulates the tensor as a linear superposition of all possible outer products of an eigenarray, a timeeigengene and a condition-eigengene, i.e., rank-1 “subtensors,” the superposition coefficients of which are tabulated in the core tensor. Each coefficient indicates the significance of the corresponding subtensor in terms of the overall information it captures in the data. PARAFAC reformulates the same data tensor into a sum of rank-1 tensor of F elements that best approximate the data tensor in a least square sense. I show that significant rank-1 subtensors can be associated with independent biological processes, which are manifested in the data tensor. Subtensors of the HOSVD capture the subprocesses: stress response, pheromone response and developmental stage. The data suggests that the conserved genes YKU70, MRE11, AIF1 and ZWF1, as well as the genes involved in the processes of retrotransposition, apoptosis and the oxidative pentose phosphate cycle may play significant, yet previously unrecognized, roles in the differential effects of HP and MD on cell cycle progression. Subtensors of PARAFAC capture the same biological processes as the 2 most significant HOSVD subtensors. A genome-wide correlation between DNA replication and initiation of RNA transcription, which is equivalent to a recently discovered correlation and might be due to a previously unknown mechanism of regulation, is independently uncovered.