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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. |
