1st Edition

Nucleic Acid Hybridization




ISBN 9781859960073
Published October 31, 1998 by Garland Science
260 Pages

USD $84.95

Prices & shipping based on shipping country


Preview

Book Description

First published in 1998. This book is targeted at the beginner who has little or no knowledge of the principles or practice of hybridization.  This text concentrates on solution and filter hybridization with a final chapter on current developments which includes DNA chips and advances in probe design.

Table of Contents

Abbreviations -- Preface -- 1. Introduction to nucleic acid hybridization -- Structure of nucleic acids -- Forces stabilizing DNA structure -- Mismatched base pairs -- Stability of nucleic acids -- Effects of pH -- Effects of temperature -- Renaturation and hybridization -- 2. Types of hybridization and uses of each method -- Solution hybridization -- Filter hybridization -- In situ hybridization -- Polymerase chain reaction -- DNA chips -- Use of more than one hybridization technique -- References -- 3. Solution hybridization: reassociation of DNA -- Reassociation kinetics -- Experimental analysis of a reassociation reaction -- Presentation of data -- Relationship between the rate of reassociation and the sequence complexity of DNA -- C0t curves of eukaryotic DNA show the presence of repeated sequences -- Determining the repetition frequency of an isolated sequence -- Isolation of different components -- Analysis of repetitive DNA -- Examining the relatedness of sequences -- Interspersion of unique and repetitive DNA -- References -- 4. Solution hybridization: RNA:DNA hybridization -- Introduction -- Progress of reaction -- DNA:RNA hybridizations with excess DNA -- DNA:RNA hybridizations in RNA excess -- Estimation of the number of genes being expressed by saturation hybridization -- Estimation of complexity of an RNA population by kinetic analysis of RNA-excess hybridization -- Detection of sequences differentially expressed in two cell types -- References -- 5. Solution hybridization: reaction conditions -- The rate of formation of duplexes -- Temperature -- Salt concentration -- Fragment length -- Other factors -- Preventing thermal breakdown of nucleic acid -- Speeding up the reaction -- Lowering the incubation temperature -- Factors affecting the stability of hybrids -- Mismatches -- References -- 6. Basic types of filter hybridization -- Probing recombinant libraries -- Southern blots -- Northern blots -- Dot blots and slot blots -- References -- 7. Hybridization strategy: long probes -- Introduction -- Factors affecting the rate of hybridization and stability -- of hybrids -- Concentration of nucleic acids -- Length of the probe -- Complexity of the probe -- Base composition -- Salt concentration -- Temperature -- Formamide -- Imperfectly matched sequences -- Hybridization accelerators -- Enzyme-linked probes -- Reaction volume -- Time of hybridization -- References -- 8. Hybridization strategy: oligonucleotide probes -- Introduction -- Factors affecting the hybridization of oligonucleotides and stability of hybrids -- Melting temperature -- Mismatches -- Rate of hybridization -- Salt concentration -- Time period for hybridization -- Hybridization accelerators -- Washing -- Control of stringency -- Optimizing conditions -- Probe design -- Length of the probe -- Single sequence probe -- Mixtures of oligonucleotide probes -- Reducing the complexity of an oligonucleotide pool -- References -- 9. Choice of probe -- Introduction -- Characteristics of probes -- DNA probes -- RNA probes -- Oligonucleotide probes -- Characterizing the probe -- Labeling the probe -- Radioactive probes: general characteristics -- Nonradioactive probes: general characteristics -- Choosing a probe -- Sensitivity required -- DNA or RNA or oligonucleotide? -- End-labeling or uniform labeling? -- References -- 10. Basic techniques: binding of nucleic acid to filters -- Types and properties of support -- Transfer of nucleic acid to filters -- Phage /colony transfer -- Transfer of DNA from gels -- Northern blots -- Dot /slot blots -- Immobilization of nucleic acid to filters -- Baking -- UV fixation -- Alkali fixation -- Hybridization in dried gels -- Experimental procedures -- Preventing degradation of RNA -- Phage /colony transfer -- Southern blots -- Northern blots -- DNA dot /slot blots -- RNA dot /slot blots -- References -- 11. Basic techniques: labeling of probes -- Introduction -- Radioactive probes -- Uniform labeling -- Labeling of DNA by random priming -- Labeling of DNA by nick translation -- Uniform labeling of RNA -- Uniform labeling by chemical methods -- End labeling of nucleic acids -- Labeling at the 5' end -- Labeling at the 3' end -- Removal of nonincorporated nucleotides -- Size exclusion -- Sephadex spin columns -- Ethanol precipitation -- Adsorption to silica particles -- Phenol extraction -- Determining the efficiency of incorporation of label -- Radioactive probes -- Nonradioactive probes -- References -- 12. Basic techniques: prehybridization, hybridization and washing -- Equipment -- Prehybridization -- Hybridization -- Washing -- Protocols for prehybridization, hybridization and washing -- References -- 13. Basic techniques: detection of hybrids -- Radioactive hybrids -- Autoradiography -- Phosphor imaging -- Detection of nonradioactive hybrids -- Chromogenic substrates -- Chemiluminescent substrates -- Enhanced chemiluminescence -- Reuse of filters and probes -- Removal of products of nonradioactive detection systems -- Stripping probe off filters -- Reuse of the probe -- References -- 14. Trouble shooting -- 15. Specific applications of techniques -- Mapping DNA -- Mapping repetitive sequences -- Detection of related sequences -- Discrimination between related sequences -- Zoo /garden blots -- Restriction fragment length polymorphisms -- DNA fingerprinting by hybridization -- Detection of mutations -- Loss of enzyme restriction sites -- Loss of genes -- Expansion of repeated sequences -- Use of allele specific oligonucleotide probes -- Reverse dot blots -- Ligase mediated gene detection -- Repeated sequences in probes -- Suppressing hybridization of repetitive sequences -- Using repetitive sequences as probes -- Semi-quantitative analyses -- Methylation -- Analysis of transcripts -- Preliminary characterization of transcripts -- Refined mapping of transcripts -- Quantitation of RNA transcripts -- Sequences expressed in one cell type and not another -- Hybrid selection of mRNA and hybrid arrest translation -- References -- 16. Current trends -- DNA arrays and chips -- Preparation of DNA arrays -- Preparation of oligonucleotide arrays -- Gene expression analyses -- DNA sequencing by hybridization -- Probes -- Developments in hybridization with oligonucleotide and peptide nucleic acid probes -- Molecular beacons -- References -- Appendices -- Appendix A: Glossary -- Appendix B: Solution hybridization equations -- Appendix C: Useful information -- Appendix D: Composition of stock solutions -- Appendix E: Suppliers -- Index.

...
View More

Author(s)

Biography

M.L.M. Anderson, Institute of Biomedical and Life Sciences, Glasgow University

Reviews

"Newcomers to biochemical research will find this handbook very useful." Aslib Book Guide Vol 64 No.5 May 99