Ribozymes and siRNA protocols

Ribozymes and siRNA protocols

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During the last few years, new discoveries in the RNA field have opened up a wealth of opportunities to specifically target mRNA for functional analysis and target validation. Contained in this volume of Methods in Molecular Biology are methods useful for the design and application of ribozymes, DNAzymes, and small interfering RNAs (siRNAs). In addition, a number of illustrations aiming to facilitate the und- standing and application of each method is included. Like any new field, ribozymes and siRNA research is rapidly evolving, and so too are the methods used to study, select, express, and control their structure and in vivo biological activities. Ribozymes and siRNA Protocols, Second Edition focuses on the latest technical advances in interfering with gene expression. It contains a short int- duction followed by general and specific protocols for using hammerhead ribozymes and derivates, DNAzymes, hairpin ribozymes, group I intron ribozymes, RNase P ribozymes, and siRNAs. Also in this volume are general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy protocols using ribozymes.
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Product details

  • Hardback | 624 pages
  • 157.5 x 233.7 x 33mm | 997.91g
  • Totowa, NJ, United States
  • English
  • Revised
  • 2nd ed. 2004
  • XVI, 624 p.
  • 1588292266
  • 9781588292261

Back cover copy

In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, hammerhead ribozymes and derivatives, and RNA interference technologies to describe in detail the exciting and successful methods now available for gene inactivation in vitro and in vivo. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms. Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.
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Table of contents

Ribozyme- and siRNA-Mediated mRNA Degradation: A General Introduction
Mouldy Sioud

Combination of Chemical and Enzymatic RNA Synthesis
Rajesh K. Gaur, Andreas Hanne, and Guido Krupp

Determination of Kinetic Parameters for Hammerhead and Hairpin Ribozymes
Martha J. Fedor

Real-Time Monitoring of RNA and DNA Reactions by Fluorescence Detection
Kumud K. Singh, Andreas Hanne, and Guido Krupp

Quantification of Ribozyme Target RNA Using Real-Time PCR
Dagmar Klein, Camillo Ricordi, and Ricardo L. Pastori

Analysis of Ribozyme Structure and Function by Nucleotide Analog Interference Mapping
Soumitra Basu, Catherine Pazsint, and Gopeswar Chowdhury

Analysis of Global Conformational Transitions in Ribozymes
David M. J. Lilley

In Vivo Detection of Ribozyme Cleavage Products and RNA Structure by Use of Terminal Transferase-Dependent PCR
Hsiu-Hua Chen, Daniela Castanotto, Jeanne LeBon, John J. Rossi, and Arthur D. Riggs

Identification of Efficient Cleavage Sites in Long-Target RNAs
Wei-Hua Pan and Gary A. Clawson

Selection In Vitro of Allosteric Ribozymes
Adam Roth and Ronald R. Breaker

Regulation of Ribozyme Cleavage Activity by Oligonucleotides
Yasuo Komatsu and Eiko Ohtsuka

Tetracycline-Regulated Expression of Hammerhead Ribozymes In Vivo
Emma T. Bowden and Anna T. Riegel

Ribozyme Expression Systems
Masayuki Sano and Kazunari Taira

Design and Expression of Chimeric U1/Ribozyme Transgenes
Roger Abounader, Robert Montgomery, Harry Dietz, and John Laterra

Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases
Jason J. Fritz, Marina Gorbatyuk, Alfred S. Lewin, and William W. Hauswirth

Helicase-Attached Novel Hybrid Ribozymes
Hiroaki Kawasaki, Masaki Warashina, Tomoko Kuwabara, and Kazunari Taira

Functional Gene Discovery Using Hybrid Ribozyme Libraries
Yoshio Kato, Masaru Tsunemi, Makoto Miyagishi, Hiroaki Kawasaki, and Kazunari Taira

Maxizyme Technology
Mayu Iyo, Hiroaki Kawasaki, and Kazunari Taira

Target-Site Selection for the 10-23 DNAzyme
Murray J. Cairns and Lun-Quan Sun

In Vitro Selected RNA-Cleaving DNA Enzymes From Combinatorial Libraries
Samitabh Chakraborti, Bandi Sriram and Akhil C. Banerjea

Nucleic Acid Sequence Analysis Using DNAzymes
Murray J. Cairns and Lun-Quan Sun

Crystallization of the Hairpin Ribozyme: Illustrative Protocols
Peter B. Rupert and Adrian R. Ferre-D'Amare

An Experimental Method for Selecting Effective Target Sites and Designing Hairpin Ribozymes
Alicia Barroso-delJesus, Elena Puerta-Fernandez, Cristina Romero-Lopez, and Alfredo Berzal-Herranz

Design and Optimization of Sequence-Specific Hairpin Ribozymes
Cristina Romero-Lopez, Alicia Barroso-delJesus, Elena Puerta-Fernandez, and Alfredo Berzal-Herranz

Design, Targeting, and Initial Screening of sTRSV-Derived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro
Max W. Richardson, Linda Hostalek, Michelle Dobson, Jason Hu, Richard Shippy, Andrew Siwkowski, Jonathan D. Marmur, Kamel Khalili, Paul E. Klotman, Arnold Hampel, and Jay Rappaport

Optimization and Application of the Group I Ribozyme Trans-Splicing Reaction
Christer Einvik, Tonje Fiskaa, Eirik W. Lundblad, and Steinar Johansen

Repair of Myotonic Dystrophy Protein Kinase (DMPK) Transcripts by Trans-Splicing Ribozymes
Leonidas A. Phylactou

General Design and Construction of RNase P Ribozymes for Gene-Targeting Application
Hua Zou, Karen Chan, Phong Trang, and Fenyong Liu

In Vitro Selection of RNase P Ribozymes That Efficiently Cleave a Target mRNA
Kihoon Kim and Fenyong Liu

In Vitro
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Review quote

"...a timely and important addition to the literature in this rapidly expanding field...The vast scope of the book will appeal to experienced researchers as a source for new avenues of study, and as a central reference for the latest technologies."


"...will be very useful for both novices and experts as a comprehensive collection of ribozyme protocols..." -Doody's Health Sciences Book Review Journal

"...will provide a valuable source of reference for many laboratories..."

-Microbiology Today
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