Single Molecule Genetic and Epigenetic Mapping of DNA in Nanofluidic Channel Arrays PDF Download
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Author: Tslil Gabrieli
Publisher:
ISBN:
Category :
Languages : en
Pages : 92
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Author: Tslil Gabrieli
Publisher:
ISBN:
Category :
Languages : en
Pages : 92
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Author: Benjamin Ryan Cipriany
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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Book Description
Microfluidics and nanofluidics have recently emerged as analytical tools for the study of biology. These devices have enabled the miniaturization of biological sample preparation and detection methods, toward consuming less sample volume and improving the sensitivity and speed of analysis. This thesis explores methods for rapid detection and sorting of individual biomolecules within a nanofluidic channel. In these devices, constructed using thin-film processing techniques, attoliter-scale volume confinement is formed to isolate individual, fluorophore-labeled biomolecules in solution for absolute quantification. These devices enable studies of the unique attributes of each molecule, often masked in ensemble-averaged measurements. Statistical sampling of many molecules is achieved by voltage-actuated, electrokinetic flow within the nanofluidic device to precisely control molecule analysis rate and achieve high throughput single molecule detection (SMD). This nanofluidic technology is applied to epigenetic analysis, enabling the study of epigenetic modifications at a single molecule level. Viable epigenetic analysis within a nanofluidic device is demonstrated using chromatin, DNA bound with histone proteins, which is shown to remain in its native state during nanofluidic confinement and electrokinetic flow under physiologically-relevant conditions. Detection of an epigenetic modification, DNA methylation, is also demonstrated to elucidate its potential for detecting multiple epigenetic marks on an individual molecule. Subsequently, an architecture for automated, high-speed sorting of individual molecules is developed. In this architecture, digital signal processing methods are implemented in a field programmable gate array to achieve real-time SMD. An electric circuit model is developed to actuate and switch electrokinetic flow of molecules, partitioning them into branches of a bifurcated nanofluidic device. An optical system for parallel SMD is realized to experimentally validate the actuation of molecule sorting in-situ. Combined, these components are utilized in automated, fluorescence-activated sorting of individual, methylated DNA molecules, which were then collected for further analysis. This device is reconfigurable and can be generalized for application to fluorescence-activated separations of other molecule types. Finally, a study of various methods for optofluidic integration is presented. The optical properties of fused-silica, silicon nitride, polydimethylsiloxane, hydrogen silisequioxane, and chemical vapor deposited oxides are investigated to consider their use in SMD applications requiring ultra-low autofluorescence and high confinement of the optical probe volume. Findings were then applied to form an optical waveguide as an fluorescence excitation source toward the dense integration of optical and nanofluidic components.
Author: Jing Tang (Ph. D.)
Publisher:
ISBN:
Category :
Languages : en
Pages : 147
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Book Description
Rapid genome characterization is one of the grand challenges of genome science today. Although the complete sequences of certain representative human genomes have been determined, genomes from a much larger number of individuals are yet to be studied in order to fully understand genome diversity and genetic diseases. While current state-of-the-art sequencing technologies are limited by the large timescale and cost required to analyze a single sample, an alternative strategy termed DNA mapping has recently received considerable attention. Unlike sequencing which produces single-base resolution, DNA mapping resolves coarse-scale (~kbp) information of the sequence, which is much faster and cheaper to obtain, but still sufficient to discern genomic differences among individuals within a given species. Advances in fluorescence microscopy have allowed the possibility to directly map a single DNA molecule. This concept, though straightforward, faces a major challenge that the entropic tendency of polymeric DNA to adopt a coiled conformation must be overcome so as to optically determine the position of specific sequences of interest on the DNA backbone. The ability to control and manipulate the conformation of single DNA molecules, especially, to stretch them into a linear format in a consistent and uniform manner, is thus crucial to the performance of such mapping devices. The focus of this thesis is to develop a reliable single DNA stretching device that can be used in single molecule DNA mapping, and to experimentally probe the fundamental physics that govern DNA deformation. In the aspect of device design, the strategy we pursue is the use of an elongational electric field with a stagnation point generated in the center of a cross-slot or T channel to stretch DNA molecules. The good compatibility of electric field with small channel dimensions allows us to use micro- or nano-fabricated channels with height on the order of or smaller than the natural size of DNA to keep the molecule always in focus, a feature desirable for any mapping applications. The presence of the stagnation point allows the possibility to dynamically trap and stretch single DNA molecules. This trapping capability ensures uniform stretching within a sample ensemble, and also allows prolonged imaging time to obtain accurate detection results. We primarily investigate the effects of channel height on the stretching process, specifically, we seek the possibility of utilizing slit-like nanoconfinement to aid DNA stretching. Although extensive previous studies have demonstrated that geometric confinement of DNA can substantially alter the conformation and dynamics of these molecules at equilibrium, no direct studies of this non-equilibrium stretching process in confinement exist prior to the work presented in this thesis. We find that slit-like confinement indeed facilitates DNA stretching by reducing the deformation Abstract rate required to achieve a certain extension. However, due to the fact that the steric interactions between the DNA and the confining walls vanish at large extensions, highly stretched DNA under confinement behaves qualitatively similar to unconfined DNA except with screened hydrodynamic interactions, and a new time scale arises that should be used to describe the large change in extension with applied deformation rate. In a consecutive study, we examine the low-extension stretching process and observe a strongly modified coil-stretch transition characterized by two distinct critical deformation rates for DNA in confinement, different from the unconfined case where a single critical deformation rate exists. With kinetic theory modeling, we demonstrate that the two-stage coilstretch transition in confinement is induced by a modified spring force law, which is essentially related to the extension-dependent steric interactions between DNA and the confining walls. We also study aspects of the equilibrium conformation and dynamics of DNA in slit-like confinement in order to provide insight into regimes where existing studies show inconsistent results. We use both experiments and simulations to demonstrate that the in-plane radius of gyration and the 3D radius of gyration of DNA behaves differently in weak confinement. In strong confinement, we do not identify any evident change in the scalings of equilibrium size, diffusivity, and longest relaxation time of the DNA with channel height from the de Gennes regime to the Odijk regime. Although the transition between the de Gennes and Odijk regimes in slit-like confinement still remains an open question, our finding adds more experimental evidence to the side of a continuous transition. The impact of this thesis will be two-fold. We design a DNA stretching device that is readily applicable to single molecule DNA mapping and establish guidelines for the effective operation of the device. Our fundamental results regarding both the equilibrium and non-equilibrium dynamics of DNA molecules in slit-like confinement will serve as a solid basis for both the design of future devices aiming to exploit confinement to manipulate biopolymers, and more complicated studies of confined polymer physics.
Author: J. Christopher Love
Publisher: John Wiley & Sons
ISBN: 3527332812
Category : Science
Languages : en
Pages : 304
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Book Description
Emphasizing their emerging capabilities, this volume provides a strong foundation for an understanding of how micro- and nanotechnologies used in biomedical research have evolved from concepts to working platforms. Volume editor Christopher Love has assembled here a highly interdisciplinary group of authors with backgrounds ranging from chemical engineering right up to materials science to reflect how the intersection of ideas from biology with engineering disciplines has spurred on innovations. In fact, a number of the basic technologies described are reaching the market to advance the discovery and development of biopharmaceuticals. The first part of the book focuses on microsystems for single-cell analysis, examining tools and techniques used to isolate cells from a range of biological samples, while the second part is dedicated to tiny technologies for modulating biological systems at the scale of individual cells, tissues or whole organisms. New tools are described which have a great potential for (pre)clinical development of interventions in a range of illnesses, such as cancer and neurological diseases. Besides describing the promising applications, the authors also highlight the ongoing challenges and opportunities in the field.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 26
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Author: Albert Jeltsch
Publisher: Springer
ISBN: 3319436244
Category : Science
Languages : en
Pages : 537
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Book Description
DNA methyltransferases are important enzymes in a broad range of organisms. Dysfunction of DNA methyltransferases in humans leads to many severe diseases, including cancer. This book focuses on the biochemical properties of these enzymes, describing their structures and mechanisms in bacteria, humans and other species, including plants, and also explains the biological processes of reading of DNA methylation and DNA demethylation. It covers many emerging aspects of the biological roles of DNA methylation functioning as an essential epigenetic mark and describes the role of DNA methylation in diseases. Moreover, the book explains modern technologies, like targeted rewriting of DNA methylation by designed DNA methyltransferases, as well as technological applications of DNA methyltransferases in DNA labelling. Finally, the book summarizes recent methods for the analysis of DNA methylation in human DNA. Overall, this book represents a comprehensive state-of-the-art- work and is a must-have for advanced researchers in the field of DNA methylation and epigenetics.
Author: Minou Bina
Publisher: Springer Science & Business Media
ISBN: 1597450979
Category : Science
Languages : en
Pages : 335
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Book Description
Completion of the sequence of the human genome represents an unpar- leled achievement in the history of biology. The project has produced nearly complete, highly accurate, and comprehensive sequences of genomes of s- eral organisms including human, mouse, drosophila, and yeast. Furthermore, the development of high-throughput technologies has led to an explosion of projects to sequence the genomes of additional organisms including rat, chimp, dog, bee, chicken, and the list is expanding. The nearly completed draft of genomic sequences from numerous species has opened a new era of research in biology and in biomedical sciences. In keeping with the interdisciplinary nature of the new scientific era, the chapters in Gene Mapping, Discovery, and Expression: Methods and Protocols recapitulate the necessity of integration of experimental and computational tools for solving - portant research problems. The general underlying theme of this volume is DNA sequence-based technologies. At one level, the book highlights the importance of databases, genome-browsers, and web-based tools for data access and ana- sis. More specifically, sequencing projects routinely deposit their data in p- licly available databases including GenBank, at the National Center of Biotechnology (NCBI) in the United States; EMBL, maintained by the European Bioinformatics Institute; and DDBJ, the DNA Data Bank of Japan. Currently, several browsers offer facile access to numerous genomic DNA sequences for gene mapping and data retrieval.
Author: Giuseppe Spoto
Publisher: Springer Science & Business Media
ISBN: 940071226X
Category : Science
Languages : en
Pages : 320
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Book Description
This book offers an overview of state-of-the-art in non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specific detection tools. Advanced materials play multiple roles in ultrasensitive detection. Optical and electrochemical detection tools are among the most widely investigated to analyze non amplified nucleic acids. Biosensors based on piezoelectric crystal have been also used to detect unamplified genomic DNA. The main scientific topics related to DNA diagnostics are discussed by an outstanding set of authors with proven experience in this field.
Author: Xiangdong Wang
Publisher: Springer
ISBN: 9401797536
Category : Medical
Languages : en
Pages : 184
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Book Description
The volume focuses on the genomics, proteomics, metabolomics, and bioinformatics of a single cell, especially lymphocytes and on understanding the molecular mechanisms of systems immunology. Based on the author’s personal experience, it provides revealing insights into the potential applications, significance, workflow, comparison, future perspectives and challenges of single-cell sequencing for identifying and developing disease-specific biomarkers in order to understand the biological function, activation and dysfunction of single cells and lymphocytes and to explore their functional roles and responses to therapies. It also provides detailed information on individual subgroups of lymphocytes, including cell characters, function, surface markers, receptor function, intracellular signals and pathways, production of inflammatory mediators, nuclear receptors and factors, omics, sequencing, disease-specific biomarkers, bioinformatics, networks and dynamic networks, their role in disease and future prospects. Dr. Xiangdong Wang is a Professor of Medicine, Director of Shanghai Institute of Clinical Bioinformatics, Director of Fudan University Center for Clinical Bioinformatics, Director of the Biomedical Research Center of Zhongshan Hospital, Deputy Director of Shanghai Respiratory Research Institute, Shanghai, China.
Author: Heinrich Klefenz
Publisher: Wiley-VCH
ISBN:
Category : Medical
Languages : de
Pages : 328
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Book Description
This volume focuses on pharmaceutical biotechnology as a key area of life sciences. The complete range of concepts, processes and technologies of biotechnology is applied in modern industrial pharmaceutical research, development and production. The results of genome sequencing and studies of biological-genetic function are combined with chemical, micro-electronic and microsystem technology to produce medical devices and diagnostic biochips. A multitude of biologically active molecules is expanded by additional novel structures created with newly arranged gene clusters and bio-catalytic chemical processes. New organisational structures in the co-operation of institutes, companies and networks enable faster knowledge and product development and immediate application of the results of research and process development. This book is the ideal source of information for scientists and engineers in research and development, for decision-makers in biotech, pharma and chemical corporations, as well as for research institutes, but also for founders of biotech companies and people working for venture capital corporations.
