Molecular Beacons Signalling Nucleic Acid Probes Methods And Protocols

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Molecular Beacons Signalling Nucleic Acid Probes Methods And Protocols

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Author : Andreas Marx
language : en
Publisher: Humana Press
Release Date : 2008-04-03

Molecular Beacons Signalling Nucleic Acid Probes Methods And Protocols written by Andreas Marx and has been published by Humana Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-04-03 with Medical categories.

From probe design to applications in clinical settings, this book provides a diverse set of instructive examples, guided by experts in the field who offer easy-to-follow experimentals. The book first offers an introduction to the basic principles of fluorescence and then describes applications of fluorogenic probes in real-time PCR, which currently is the gold standard for quantitative DNA and RNA analysis. Coverage extends the potential of realtime as well as advocates simplifications of the probe technologies. It also presents a new simplified molecular beacon design, EasyBeacons, and demonstrates the utility in DNA methylation profiling.

Molecular Beacons Signalling Nucleic Acid Probes Methods And Protocols

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Author : Andreas Marx
language : en
Publisher: Humana Press
Release Date : 2008-04-03

Molecular Beacons Signalling Nucleic Acid Probes Methods And Protocols written by Andreas Marx and has been published by Humana Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-04-03 with Medical categories.

From probe design to applications in clinical settings, this book provides a diverse set of instructive examples, guided by experts in the field who offer easy-to-follow experimentals. The book first offers an introduction to the basic principles of fluorescence and then describes applications of fluorogenic probes in real-time PCR, which currently is the gold standard for quantitative DNA and RNA analysis. Coverage extends the potential of realtime as well as advocates simplifications of the probe technologies. It also presents a new simplified molecular beacon design, EasyBeacons, and demonstrates the utility in DNA methylation profiling.

Molecular Beacons

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Author : Chaoyong James Yang
language : en
Publisher: Springer Science & Business Media
Release Date : 2013-08-15

Molecular Beacons written by Chaoyong James Yang and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2013-08-15 with Science categories.

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.

Hedgehog Signaling Protocols

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Author : Jamila I. Horabin
language : en
Publisher: Springer Science & Business Media
Release Date : 2007-09-13

Hedgehog Signaling Protocols written by Jamila I. Horabin and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2007-09-13 with Medical categories.

In both vertebrates and invertebrates, Hedgehog signaling regulates the development of the tissues and structures of the body. In addition, many cancers and diseases arise as a result of misregulation of the pathway, which makes it an incredibly important system to understand. This volume examines various methods used in the analysis of the Hedgehog signaling pathway, bringing together different lines of analyses into one accessible and comprehensive text. This volume provides a single source for investigators, presenting several different experimental approaches, which are all varied and broad in nature, as well as procedures for a variety of different model systems. Researchers will find this volume to be an invaluble tool as they work towards deeper understanding of the underlying molecular events that the Hedgehog signaling pathway regulates.

Nucleic Acid Probes

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Author : Robert H. Symons
language : en
Publisher: CRC Press
Release Date : 1989-03-31

Nucleic Acid Probes written by Robert H. Symons and has been published by CRC Press this book supported file pdf, txt, epub, kindle and other format this book has been release on 1989-03-31 with Medical categories.

With each chapter written by a distinguished expert in the biochemistry field, this comprehensive volume describes the preparation and use of a variety of radioactive and non-radioactive probes in situations ranging from research laboratories to routine diagnosis laboratories. The enzymatic and chemical techniques for labeling nucleic acid probes with radioisotopes and with non-radioactive ligands and haptens are discussed. Additionally, the associated methods for their detection, the use of these probes in the diagnosis of human and microbial pathogens, of plant viruses and viroids, and of human genetic disorders, as well as in the detection of nucleic acids in tissues and cells by in situ hybridization are presented.

Mediator Displacement Lamp A Novel Method For Universal Sequence Specific Detection Of Isothermal Nucleic Acid Amplification

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Author : Lisa Becherer
language : en
Publisher:
Release Date : 2020

Mediator Displacement Lamp A Novel Method For Universal Sequence Specific Detection Of Isothermal Nucleic Acid Amplification written by Lisa Becherer and has been published by this book supported file pdf, txt, epub, kindle and other format this book has been release on 2020 with categories.

Abstract: Nucleic acid amplification tests (NAATs) are an essential diagnostic tool throughout the field of life sciences, including clinical applications, food quality control, and environmental monitoring. In the last 20 years, polymerase chain reaction (PCR) has been steadily replaced by isothermal alternatives. These are especially suitable for point-of-need (PON) applications. Loop-mediated isothermal amplification (LAMP) stands out as a particularly robust and highly sensitive method. Special attention is given to the sequence-specific, fluorescence-based detection of LAMP, which allows the highly specific and simultaneous detection of various targets (multiplexing). However, current state-of-the-art methods suffer from complex probe design and elaborate optimization work, which is required for the detection of different targets due to the use of target-specific fluorogenic probes. The principal objectives of this thesis are first, to develop an improved sequence-specific detection method for LAMP with a simplified probe design, second, to verify its analytical performance and third, to validate the method by using it to analyze clinical samples. Furthermore, the feasibility of transferring the novel method to two other platforms, digital nucleic acid testing and electrochemical nucleic acid testing, is investigated. A novel method was successfully developed and named mediator displacement (MD) LAMP. MD LAMP stands out from other state-of-the-art methods because of its use of unique, target-independent fluorogenic reporter molecules. The working principle involves a non-fluorogenic MD probe which features a primer bound to a generic mediator. Mediator displacement occurs during the amplification of target DNA. Fluorescence signal generation is then induced by the interaction between the displaced mediator and a universal reporter molecule. The universal mediator-reporter set can be used to detect various targets. Simplified probe design was demonstrated by the example of a reverse transcription (RT)-LAMP of human immunodeficiency virus (HIV)-1 RNA. The time required for MD probe design was first compared with the time needed to create a state-of-the-art molecular beacon (MD: 10 minutes, molecular beacon: 3-4 hours). Moreover, HIV-1 MD RT-LAMP surpassed molecular beacon-based HIV-1 RT-LAMP, with times to positive 4.1 ± 0.1 minutes shorter (16-20 minutes for 10^3-10^6 HIV RNA copies per reaction) and double the signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, molecular beacon: 2.7 ± 0.4; n = 15). The analytical performance parameters were equally good for both detection methods (limit of detection for MD: 132 HIV RNA copies per reaction, molecular beacon: 141 HIV RNA copies per reaction; linearity for MD and molecular beacon: R^2 = 0.94). Furthermore, multiplex detection of two different targets was demonstrated in a biplex MD RT-LAMP for the simultaneous detection of HIV-1 and human T-lymphotropic virus (HTLV)-1. To present an additional possible application, the biplex RT-LAMP of HIV-1 and HTLV-1 was transferred to a centrifugal microfluidic platform for digital nucleic acid amplification in droplets. Digital NAATs allow precise quantification without the need for standard curves. Compared to existing viral load measurements, the novel assay excelled through the simultaneous and quantitative detection of relevant coinfections. The universal mediator-reporter sets were successfully used for the detection of a second clinically relevant target panel, which is associated with yaws, a neglected tropical disease. For the molecular detection of yaws, a biplex MD LAMP of Treponema pallidum and Haemophilus ducreyi (TPHD-LAMP) was designed, optimized and clinically validated with 293 patient samples. The samples originated from individuals with yaws-like skin lesions in Ghana and Papua New Guinea. So far, no isothermal alternatives to PCR which enable differentiation between T. pallidum, which causes yaws, and H. ducreyi, which is responsible for skin lesions of similar appearance, are available. TPHD-LAMP revealed high diagnostic sensitivities and specificities for T. pallidum (84.7 % and 95.7 %) and H. ducreyi (91.6 % and 84.8 %) compared to TaqMan singleplex qPCR. TPHD-LAMP has become the focus of a large clinical trial conducted in three yaws endemic African countries and linked to the goal of supporting national yaws eradication programmes. So far, the analytical performance of MD LAMP has been verified, the feasibility of a digital MD RT-LAMP has been shown, and MD LAMP has been clinically validated with patient samples. To emphasize the versatility of this novel method, its compatibility with real-time electrochemical NAATs was also demonstrated. Microarray-based electrochemical detection facilitates the simultaneous detection of multiple targets by using spatially-resolved measurement with the help of immobilized probes. In addition, it enables the use of very small and therefore portable devices. To exploit these benefits, MD LAMP was adapted to electrochemical detection by immobilizing universal reporter molecules on an electrode microarray. The feasibility of electrochemical MD LAMP was demonstrated for real-time detection of T. pallidum and end-point detection of HIV-1. Rapid signal increase was observed in real time after 15 minutes for 1000 T. pallidum DNA copies per reaction. Electrochemical MD LAMP enables the use of universal electrode microarrays and could thus bring economic advantages with regard to large-scale fabrication. This thesis concludes with a critical review of the methods and the method-related instrumentation for the sequence-specific detection of LAMP, which are based on a diverse variety of sensing techniques. The multitude of methods is systematically classified and critically evaluated according to a catalogue of criteria covering analytical performance, handling of complex samples, multiplexing, and quantification. The most widespread sensing technique is based on fluorescence detection and is used in around half of the discussed methods. A particular highlight is the universal character of a few fluorescence-based methods, which use generic probes applicable to different targets

Neuroinformatics

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Author : Chiquito J. Crasto
language : en
Publisher: Springer Science & Business Media
Release Date : 2007-11-29

Neuroinformatics written by Chiquito J. Crasto and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2007-11-29 with Medical categories.

Neuroinformatics presents cutting-edge techniques for the synergistic study of neuroinformatics. The book facilitates the efforts of discovering neuroscience through the sharing of data and the use of computational models. It demonstrates the use of neuroinformatic components as a mechanism for understanding complex disorders. It contains detailed explanations, advantages, and disadvantages of traditional and non-invasive imaging methods.

The Nucleic Acid Protocols Handbook

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Author : Ralph Rapley
language : en
Publisher: Springer Science & Business Media
Release Date : 2000-03-24

The Nucleic Acid Protocols Handbook written by Ralph Rapley and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2000-03-24 with Science categories.

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 BiologyTM 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.

Affinity Chromatography

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Author : Michael Zachariou
language : en
Publisher: Springer Science & Business Media
Release Date : 2008

Affinity Chromatography written by Michael Zachariou and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008 with Medical categories.

Thirty-eight years after its introduction, affinity chromatography remains a key tool in the armory of separation techniques available to separation and interaction scientists. Expanded and updated from the first edition, Affinity Chromatography: Methods and Protocols, Second Edition, provides the beginner with the practical knowledge needed to develop affinity separations suitable for a variety of applications relevant to the post-genomic era. This second edition expands on the first edition by introducing more state-of-the-art protocols used in affinity chromatography. This new edition also describes protocols that demonstrate the concept of affinity chromatography being applied to meet the modern high throughput screening demands of researchers and development scientists whilst expanding on some more traditional affinity chromatography approaches that have become of greater interest to separation scientists. Chapters in this cutting-edge text expand on affinity chromatography techniques that currently enjoy frequent citation in the literature from those purifying biomolecules. Other chapters include protocols describing the use of a variety of fusion tags as well as how to cleave them, so as to allow the scientists to study the native phenotype of the protein. Renowned researchers also include protocols detailing diverse applications of affinity chromatography such as its use in catalytic reactions, DNA purification, whole cell separations and for the isolation of phosphorylated proteins. Affinity Chromatography: Methods and Protocols, Second Edition, is an essential reference for those interested in separation sciences, particularly in the pharmaceutical and biological research sectors, that have an interest in isolating macromolecules rapidly, quantitatively, and with high purity.

Hematopoietic Stem Cell Protocols

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Author : Kevin D. Bunting
language : en
Publisher: Springer Science & Business Media
Release Date : 2008-01-31

Hematopoietic Stem Cell Protocols written by Kevin D. Bunting and has been published by Springer Science & Business Media this book supported file pdf, txt, epub, kindle and other format this book has been release on 2008-01-31 with Science categories.

This revised edition provides up-to-date protocols developed in the HSC field. A team of leading researchers supply this volume with in-depth, readily reproducible methods for effective characterization of HSC and their developmental potential. The book provides detailed flow cytometry protocols for thorough analysis of enriched HSC populations, and offers a variety of transplantation approaches to measure HSC function in vivo. This is a much needed technical resource in the critically important field of stem cell investigation.