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Author: Alexandre Louis Andre Persat
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Microfluidics has recently enabled new capabilities in life sciences research. By leveraging physics at the microscale, novel miniaturized methods and devices provide fundamental improvements over traditional assays including higher sensitivity, massive parallelization, speed and automation. For example, nucleic acid analyses such as PCR or capillary electrophoresis are now commonly executed on microfluidic platforms. However, extracting and isolating a set of molecules of interest from a biological sample remains a widespread challenge, in particular when the target is a nucleic acid; such sample preparation has been identified as the "weak link" of microfluidics. Moreover, the discovery of classes of small RNA such as microRNA (miRNA) has revealed the limitations of benchtop preparation methods. This work tackles these issues by leveraging an electrophoretic focusing method, isotachophoresis (ITP), to perform selective focusing of specific nucleic acid molecules contained in complex mixtures. This includes extraction of genomic DNA from blood and isolation of miRNA from total RNA. Also, we leverage the unique physics of ITP to perform simultaneous purification and analysis of these molecules, thus enabling automated analysis at unprecedented speed. ITP is a robust electrophoretic preconcentration technique which generates strong electric field gradients, and enables selective focusing and separation of charged species based on their electrophoretic mobilities. In this work, we show that we can extract and purify genomic DNA from chemically lysed whole blood samples by carefully controlling ITP electrolyte chemistry to achieve selective focusing of genetic material. We show that ITP outputs PCR-compatible DNA with high efficiency in about 1 min. This novel sample preparation technique allows for efficient, fast, and automated purification of DNA from 10 nL to 1 [Mu]L of biological fluids. We also demonstrate that ITP focusing of microRNA -- short (~22 nt), non-coding RNA regulating gene expression -- enables quantification and sequence specific detection. We leverage both the selective and preconcentration capability of ITP for the quantification of global miRNA abundance. This allows for the measurement of RNA silencing activity in specific cells or tissues. We have optimized ITP chemistry and used a multi-stage injection strategy to selectively preconcentrate and quantify RNA shorter than 40 nt. We discuss results of miRNA quantification for a wide variety of samples. Additionally, we show that the combination of selective ITP focusing with simultaneous hybridization with molecular beacons is an efficient method for detection and quantitation of specific miRNA sequences. We demonstrate the efficacy of this assay for the detection of a liver specific miRNA. Finally, we show that we can use ITP to perform polymerase chain reaction in isothermal conditions by creating a cycles of chemicals mimicking thermal cycling.
Author: Alexandre Louis Andre Persat
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Microfluidics has recently enabled new capabilities in life sciences research. By leveraging physics at the microscale, novel miniaturized methods and devices provide fundamental improvements over traditional assays including higher sensitivity, massive parallelization, speed and automation. For example, nucleic acid analyses such as PCR or capillary electrophoresis are now commonly executed on microfluidic platforms. However, extracting and isolating a set of molecules of interest from a biological sample remains a widespread challenge, in particular when the target is a nucleic acid; such sample preparation has been identified as the "weak link" of microfluidics. Moreover, the discovery of classes of small RNA such as microRNA (miRNA) has revealed the limitations of benchtop preparation methods. This work tackles these issues by leveraging an electrophoretic focusing method, isotachophoresis (ITP), to perform selective focusing of specific nucleic acid molecules contained in complex mixtures. This includes extraction of genomic DNA from blood and isolation of miRNA from total RNA. Also, we leverage the unique physics of ITP to perform simultaneous purification and analysis of these molecules, thus enabling automated analysis at unprecedented speed. ITP is a robust electrophoretic preconcentration technique which generates strong electric field gradients, and enables selective focusing and separation of charged species based on their electrophoretic mobilities. In this work, we show that we can extract and purify genomic DNA from chemically lysed whole blood samples by carefully controlling ITP electrolyte chemistry to achieve selective focusing of genetic material. We show that ITP outputs PCR-compatible DNA with high efficiency in about 1 min. This novel sample preparation technique allows for efficient, fast, and automated purification of DNA from 10 nL to 1 [Mu]L of biological fluids. We also demonstrate that ITP focusing of microRNA -- short (~22 nt), non-coding RNA regulating gene expression -- enables quantification and sequence specific detection. We leverage both the selective and preconcentration capability of ITP for the quantification of global miRNA abundance. This allows for the measurement of RNA silencing activity in specific cells or tissues. We have optimized ITP chemistry and used a multi-stage injection strategy to selectively preconcentrate and quantify RNA shorter than 40 nt. We discuss results of miRNA quantification for a wide variety of samples. Additionally, we show that the combination of selective ITP focusing with simultaneous hybridization with molecular beacons is an efficient method for detection and quantitation of specific miRNA sequences. We demonstrate the efficacy of this assay for the detection of a liver specific miRNA. Finally, we show that we can use ITP to perform polymerase chain reaction in isothermal conditions by creating a cycles of chemicals mimicking thermal cycling.
Author: Dongyou Liu
Publisher: CRC Press
ISBN: 1420070975
Category : Science
Languages : en
Pages : 572
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Book Description
An Indispensable Roadmap for Nucleic Acid Preparation Although Friedrich Miescher described the first isolation of nucleic acid in 1869, it was not until 1953 that James Watson and Francis Crick successfully deciphered the structural basis of DNA duplex. Needless to say, in the years since, enormous advances have been made in the study of nucleic a
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: Jacquie T. Keer
Publisher: Royal Society of Chemistry
ISBN: 0854043675
Category : Science
Languages : en
Pages : 274
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Book Description
An indispensable handbook of the highest standard for those working in the fields of food analysis and forensic applications.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 12
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Book Description
Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in an inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing electrolyte ions. Conversely, the trailing electrolyte ions have a slow electrophoretic mobility, so they lag behind the sample, thus trapping the species of interest between the LE and TE streams. In a typical isotachophoresis configuration, the electric field is applied in a direction parallel to the direction of flow. The species then form bands that stretch across the width of the channel. A major limitation of that approach is that only a finite amount of sample can be processed at once, and the sample must be processed in batches. For our purposes, a form of free-flow isotachophoresis is more convenient, where the DNA forms a band parallel to the edges of the channel. To achieve this, in our chip, the electric field is applied transversely. This creates a force perpendicular to the direction of flow, which causes the different ions to migrate across the flow direction. Because the mobility of the DNA is between the mobility of the leading and the trailing electrolyte, the DNA is focused in a tight band near the center of the channel. The stream of DNA can then be directed to a different output to produce a highly concentrated outlet stream without batch processing. One hurdle that must be overcome for successful ITP is isolating the electrochemical reactions that result from the application of high voltage for the actual process of isotachophoresis. The electrochemical reactions that occur around metal electrodes produce bubbles and pH changes that are detrimental to successful ITP. The design of the chips we use incorporates polyacrylamide gels to serve as electrodes along the central channel. For our design, the metal electrodes are located away from the chip, and high conductivity buffer streams carry the potential to the chip, functioning as a 'liquid electrode.' The stream then runs alongside a gel barrier. The gel electrode permits ion transfer while simultaneously isolating the separation chamber from any contaminants in the outer, 'liquid electrode' streams. The difference in potential from one side of the chip to the other creates an electric field. This field traverses the inner, separation channel, containing the leading electrolyte, the trailing electrolyte, and the sample of interest (DNA). To increase the ease of use of the chips, a newer chip design has been fabricated. This design has wire electrodes integrated on the chip, rather than elsewhere. To keep the pH changes and bubbling isolated from the separation channel, the chip contains deeper wells near the electrodes so that the flowing buffer can wash away any gases that form around the electrode. This design is significantly more compact because it eliminates the cumbersome electrode boxes. Eliminating the electrode boxes also decreases the required voltage, making the experiments safer. This happens because when the 'liquid electrode' streams travel through small diameter tubing, they lose much of their voltage due to the electrical resistance of the fluid in the tubing.
Author: Ralph Rapley
Publisher: Springer Science & Business Media
ISBN: 1592590381
Category : Science
Languages : en
Pages : 997
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Book Description
A comprehensive treasury of all the key molecular biology methods-ranging from DNA extraction to gene localization in situ-needed to function effectively in the modern laboratory. Each of the 120 highly successful techniques follows the format of the much acclaimed Methods in Molecular BiologyOao series, providing an introduction to the scientific basis of each technique, a complete listing of all the necessary materials and reagents, and clear step-by-step instruction to permit error-free execution. Included for each technique are notes about pitfalls to avoid, troubleshooting tips, alternate methods, and explanations of the reasons for certain steps-all key elements contributing significantly to success or failure in the lab. The Nucleic Acid Protocols Handbook constitutes today's most comprehensive collection of all the key classic and cutting-edge techniques for the successful isolation, analysis, and manipulation of nucleic acids by both experienced researchers and those new to the field."
Author: Botho Bowien
Publisher: American Scientific Publishers
ISBN:
Category : Science
Languages : en
Pages : 208
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Book Description
Although it's stated only on the back cover, this book seems to initiate a new series (from this new publisher) titled "Genome Science and Nucleic Acids." Ten review chapters address the stabilization of nucleic acids prior to isolation; isolation of genomic DNA, and of plasmid DNA; purification of plasmid DNA, and of viral nucleic acids; isolation of RNA; DNA cleanup from gels, PCR, sequencing, and others; nucleic acids of pathogens from biological samples for real-time PCR analysis; extraction of DNA from paraffin embedded bone marrow trephine biopsies; and DNA and forensic applications. Nineteen of the 20 contributors to the present work are based in Germany (one is in the UK); their work was brought together by editors Bowien (microbiology and genetics, U. of Gottingen) and Durre (microbiology and biotechnology, U. of Ulm). Annotation (c)2003 Book News, Inc., Portland, OR (booknews.com).
Author: Petr Boček
Publisher:
ISBN:
Category : Electrophoresis
Languages : en
Pages : 268
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Book Description
Author: Christopher J. Holloway
Publisher:
ISBN:
Category : Science
Languages : en
Pages : 428
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Book Description
Author: Christoph Heller
Publisher: Springer Science & Business Media
ISBN: 3322910156
Category : Technology & Engineering
Languages : en
Pages : 318
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Book Description
Die Kapillarelektrophorese (CE) ist heute in biochemischen Laboratorien noch nicht als Routinemethode zur DNA-Trennung etabliert, obwohl sie diverse Vorzüge gegenüber den derzeitigen Methoden besitzt. In diesem Band stellen verschiedene Wissenschaftler die theoretischen Aspekte der Trennung von Oligonucleinsäuren mit der CE, die instrumentellen Grundlagen und deren Grenzen und schließlich die praktische Anwendung in der Biochemie und Molekularbiologie vor. This book on capillary electrophoresis is unique in its focus on the separation of nucleic acids. The importance of electrophoretic separation in every molecular biology laboratory justifies this specialization, which is also reflected in the development of instrumentation. This book is aimed to help to implement this rather new and promising technology in the biological laboratories and to help to overcome typical problems that can occur when starting with a new technique. It should also trigger further development in the field. It covers the theoretical background as well as practical examples of usual applications. The authors are all experts in their field, having many years of experience with capillary electrophoresis and their application to nucleic acids.
