Designing Automated Systems for Sample Preparation of Nucleic Acids Using Isotachophoresis PDF Download
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Author: Lewis A. Marshall
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Purified DNA serves as a template for a wide array of analysis techniques, ranging from sequencing to PCR and hybridization assays. DNA analysis can be used for clinical diagnosis, for forensic investigation, and for a range of research purposes. These analysis techniques improve each year, but they are all constrained by the availability of purified DNA. DNA is typically derived from raw biological samples that contain a host of other molecular species, including proteins, lipids and metal ions. These species can inhibit analysis of the DNA, so purification of DNA from complex sample matrices is a necessary precursor to analysis. Typically, DNA purification is performed using either liquid-liquid extraction or solid-phase extraction, both of which require manual labor, involve toxic chemicals, and are difficult to miniaturize. Isotachophoresis (ITP) is an alternative method for DNA purification that does not rely on specialized surface chemistry or toxic chemical species. Instead, ITP uses electric fields to selectively pre-concentrate DNA from a raw sample, and simultaneously separate it from inhibiting species. ITP purification of DNA has been demonstrated from human serum, plasma, and whole blood, and the same technique has been used to purify RNA from bacteria in human blood and urine. Until recently, the parameters governing extraction efficiency, throughput, and separation quality in ITP purification were not well established. This thesis is focused on rational analysis for designing and optimizing ITP systems for rapid, high quality DNA purification.
Author: Lewis A. Marshall
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Purified DNA serves as a template for a wide array of analysis techniques, ranging from sequencing to PCR and hybridization assays. DNA analysis can be used for clinical diagnosis, for forensic investigation, and for a range of research purposes. These analysis techniques improve each year, but they are all constrained by the availability of purified DNA. DNA is typically derived from raw biological samples that contain a host of other molecular species, including proteins, lipids and metal ions. These species can inhibit analysis of the DNA, so purification of DNA from complex sample matrices is a necessary precursor to analysis. Typically, DNA purification is performed using either liquid-liquid extraction or solid-phase extraction, both of which require manual labor, involve toxic chemicals, and are difficult to miniaturize. Isotachophoresis (ITP) is an alternative method for DNA purification that does not rely on specialized surface chemistry or toxic chemical species. Instead, ITP uses electric fields to selectively pre-concentrate DNA from a raw sample, and simultaneously separate it from inhibiting species. ITP purification of DNA has been demonstrated from human serum, plasma, and whole blood, and the same technique has been used to purify RNA from bacteria in human blood and urine. Until recently, the parameters governing extraction efficiency, throughput, and separation quality in ITP purification were not well established. This thesis is focused on rational analysis for designing and optimizing ITP systems for rapid, high quality DNA purification.
Author: John Glenn Minson
Publisher:
ISBN:
Category : Microfluidic devices
Languages : en
Pages : 58
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Publisher: Academic Press
ISBN: 0323853048
Category : Science
Languages : en
Pages : 370
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Book Description
Micro/Nanofluidics and Lab-on-Chip Based Emerging Technologies for Biomedical and Translational Research Applications - Part B, Volume 187 represents the collation of chapters written by eminent scientists worldwide. Chapters in this new release include Design and fabrication of microfluidics devices for molecular biology applications, Micro/Nanofluidics devices for drug delivery, From organ-on-chip to body-on-chip: the next generation of microfluidics platforms for in vitro drug toxicity testing, Micro/Nanofluidics for high throughput drug screening, Design, fabrication and assembly of lab-on-a-chip and its uses, Advances in microfluidic 3D cell culture for pre-clinical drug development, Tissue and organ culture on lab-on-a chip for biomedical applications, and much more. - Offers a basic understanding of the state-of-the-art design and fabrication of microfluidics/ nanofluidics and lab on chip - Explains how to develop microfluidics/nanofluidic for advanced application such as healthcare, high throughout drug screening, 3D cell culture and organ-on-chip - Discusses the emerging demands and research of micro/nanofluidic based devices in biomedical and translational research applications
Author: Anita Rogacs
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
This dissertation describes three techniques aimed at automated, sensitive sample preparation and detection of RNA from complex samples. Microfluidic systems have advanced the state of the art for a wide number of chemical and biological assays, but robust and efficient sample preparation remains a major challenge. One of the most difficult processes is the lysing, purification, and detection of target RNA from whole blood samples. This dissertation addresses key challenges in each of these RNA workflow phases. In the first part of the dissertation, we demonstrate a novel assay for lysing followed by physicochemical extraction and isotachophoresis-based purification of 16S ribosomal RNA from whole human blood infected with Pseudomonas Putida. This assay is unique in that the extraction can be automated on-chip using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (like blood), and produces a purified total nucleic acid (NA) sample which is compatible with enzymatic amplification assays. We show that the purified RNA are compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR. In the second part of the dissertation, we present a model to aid in design and optimization of a wide range of electrophoresis (including isotachophoresis) assays. Our model captures the important contributors to the effects of temperature on the observable electrophoretic mobilities of small ions, and on solution ionic strength, conductivity and pH; the most relevant parameters in molecular reactions and separation assays. Our temperature model includes relations for temperature-dependent viscosity, ionic strength corrections, degree of ionization (pK), and ion solvation effects on mobility. We incorporate thermophysical data for water viscosity; temperature-dependence of the Onsager-Fuoss model for finite ionic strength effects on mobility; temperature-dependence of the extended Debye-Huckel theory for correction of ionic activity; the Clarke-Glew approach and tabulated thermodynamic quantities of ionization reaction for acid dissociation constants as a function of temperature; and species-specific, empirically evaluated correction terms for temperature-dependence of Stokes' radii. We incorporated our model into a MATLAB-based simulation tool we named Simulation of Temperature Effects on ElectroPhoresis (STEEP). We validated our model using conductance and pH measurements across a temperature variation of 25°C to 70°C for a set of electrolytes routinely used in electrophoresis. The model accurately captures electrolyte solution pH and conductivity, including important effects not captured by simple Walden type relations. In the third and final part of the dissertation, we introduce a cost-effective and simple-to-implement method for direct detection of RNA, by analyzing images of randomly distributed multicolor fluorescent beads bound by the target molecule. We term our method particle imaging, tracking and collocation (PITC). We use a fairly standard epifluorescence microscopy setup fitted with an off-the-shelf dual view color separator attachment which images fluorescence emission at two wavelengths onto two respective halves of a single CCD image array. We perform automated analysis of these two color channels to track thousands of particle images in either or both wavelengths, and we track particle image motion in time and space. We can quantify particle image wavelength (or wavelength ratio), absolute intensities, and particle image diameter. We also perform cross-correlation image analyses on this multi-wavelength data to track particle collocations and the degree of correlation of particle motion. Particles or cells can be suspended in solution and flowed through a wide variety of microchannels (with optical access for image collection). Particles can be transported through the detection region via electrophoresis and/or pressure driven flow, to increase throughput of analysis. We here introduce and evaluate the performance of our method. We use Monte Carlo simulations to demonstrate robustness of the algorithm and optimize algorithm parameters. We present an experimental demonstration of the method on challenging image data, including flow of randomly distributed Brownian particles and particle populations which undergo particle-to-particle binding. We show results of bead collocation measurements on bead-to-bead binding created by the E.coli 16S rRNA gene.
Author: Danny van Noort
Publisher: MDPI
ISBN: 3039215620
Category : Technology & Engineering
Languages : en
Pages : 200
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Book Description
Microfluidics-based devices play an important role in creating realistic microenvironments in which cell cultures can thrive. They can, for example, be used to monitor drug toxicity and perform medical diagnostics, and be in a static-, perfusion- or droplet-based device. They can also be used to study cell-cell, cell-matrix or cell-surface interactions. Cells can be either single cells, 3D cell cultures or co-cultures. Other organisms could include bacteria, zebra fish embryo, C. elegans, to name a few.
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 2164
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Book Description
Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.
Author: Danny L. Wiedbrauk
Publisher: Elsevier
ISBN: 0080536891
Category : Science
Languages : en
Pages : 405
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Book Description
Molecular diagnostic procedures have been described in a number of recent books and articles. However, these publications have not focused on virus detection, nor have they provided practical protocols for the newer molecular methods. Written by the inventors or principal developers of these technologies, Molecular Methods for Virus Detection provides both reviews of individual methods and instructions for detecting virus nucleic acid sequences in clinical specimens. Each procedure includes quality assurance protocols that are often ignored by other methodology books. Molecular Methods for Virus Detection provides clinically relevant procedures for many of the newer diagnostic methodologies. - Provides state-of-the-art PCR methods for amplification, quantitation, in situ hybridization, and multiplex reactions - Goes beyond PCR with protocols for 3SR, NASBA, LCR, SDA, and LAT - Covers important virus detection methods such as in situ hybridization; Southern, dot, and slot blots; branched chain signal amplification; and chemiluminescence - Includes quality control information crucial in research and clinical laboratories - Most chapters are written by the inventors and principal developers of the methodologies - Includes color plates, 77 figures, and 18 tables
Author: Chaoyong James Yang
Publisher: Springer Science & Business Media
ISBN: 3642391095
Category : Science
Languages : en
Pages : 201
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Book Description
Molecular Beacons explains working principle of molecular beacons, discusses their design, synthesis, purification and characterization, explores their thermodynamic and kinetic properties, and more importantly, reviews their in vivo and in vitro applications with the emphasis on the design and modification of molecular beacons for in vivo mRNA imaging applications. This book is designed to bring together in a single resource an organized and comprehensive view of molecular beacons and will be a valuable resource for academic, clinical and industrial scientists and graduate students who may consider exploring molecular beacons in their research or practice. Chaoyong James Yang is the Lu Jiaxi Professor of Chemistry at Xiamen University, China. Weihong Tan is a Distinguished Professor of Chemistry and Biomedical Engineering at Hunan University, China and also a University of Florida Distinguished Professor and V. T. and Louis Jackson Professor of Chemistry at the University of Florida, USA.
Author:
Publisher:
ISBN: 9781562385514
Category : Pathological laboratories
Languages : en
Pages : 39
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Book Description
Author: F.M. Everaerts
Publisher: Elsevier
ISBN: 0080858066
Category : Science
Languages : en
Pages : 433
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Book Description
Isotachophoresis
