M L M Anderson

Nucleic Acid Hybridization

• Broschiertes Buch

Produktbeschreibung

Simple practical guide which contains all relevant background information;; Deals with the applications of nucleic acid hybridization in detail;; Provides comprehensive trouble-shooting advice;; This is an important practical guide which describes wha

Produktdetails

• Verlag: CRC Press
• Seitenzahl: 260
• Erscheinungstermin: 15. Juni 1998
• Englisch
• Abmessung: 234mm x 156mm x 14mm
• Gewicht: 367g
• ISBN-13: 9781859960073
• ISBN-10: 1859960073
• Artikelnr.: 27019146

Autorenporträt

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

Inhaltsangabe

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.