Mutation detection : a practical approach / edited by R.G.H. Cotton, E. Edkins, and S. Forrest.

Includes bibliographical references and index.

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Mutation detection is increasingly undertaken in a wide spectrum of research areas: in medicine it is fundamental in isolating disease genes and diagnosis, and is especially important in cancer research; in biology, commercially important genes can be identified by the mutations they contain. But mutation detection is time-consuming and expensive. This volume offers tried and tested protocols for a range of detection methods, from the labs of researchers in the field.

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Cover -- Contents -- List of Contributors -- Abbreviations -- Introduction -- References -- 1. Single-strand conformation polymorphism analysis -- 1. Introduction -- 2. PCR-SSCP using polyacrylamide slab gel -- PCR Optimization and primer design -- Pre-amplification and isolation by agarose gel electrophoresis -- PCR using [[Sup(32)]P]deoxynucleotide triphosphate -- Removal of 3' appendage -- SSCP gel electrophoresis -- Interpretation of autoradiogram -- Re-amplification and direct sequencing -- Gel matrices other than polyacrylamide.

Restriction endonuclease fingerprinting and dideoxy fingerprinting -- 3. Fluorescent SSCP in an automated DNA sequencer -- Primer design in post-PCR fluorescent labelling -- Fluorescent labelling by 3' exchange reaction -- SSCP in capillary electrophoresis (CE-SSCP) -- Data processing -- Acknowledgements -- References -- 2. Single-stranded conformation polymorphism and heteroduplex analysis -- 1. Introduction -- 2. Optimization of the PCR reaction -- 3. SSCP sample prepration -- 4. Optimization of SSCP/HA detection -- 5. Multiplexing -- 6. Interpretation of results -- 7. Applications.

8. Other methods -- References -- 3. Comprehensive mutation detection with denaturing gradient gel electrophoresis -- 1. Introduction -- The scope of DGGE, its distinctive capabilities, and the nature of results -- 2. Background -- 3. Basic principle, the physical properties of DNA -- 4. Overview of the procedures in searching for mutants -- Defining segments for scrutiny -- Sample preparation -- Gradient and velocity separations -- Features of the gel patterns -- Discrimination of zygozygosity -- Comments -- 5. Use of the psoralen cross-link as a clamp -- The psoralen protocol.

6. Computational tools -- What is a meltmap? -- Meltmap protocol -- Predicting electrophoretic separations -- Computer operations for MUTRAV -- 7. Other members of the DGGE family -- Gel separations in a uniform, partially denaturing environment -- Capillary electrophoresis -- The thermal gradient -- The temperature ramp -- 2D length and gradient separations -- 8. End notes -- Acknowledgments -- References -- 4. Cleavage using RNase to detect mutations -- 1. Introduction -- 2. RNase protection assay for mutation detection -- Evaluation of the sensitivity -- Source material.

PCR for RNase protection assay -- RNA probe preparation -- RNase protection -- Detection of digested probe -- Mutation detection by sequencing of the PCR products -- Other modified methodologies for mutation detection -- Acknowledgements -- References -- 5. Cleavage of mismatched bases using chemical reagents -- 1. Introduction -- 2. Basic procedures -- Comments on the basic procedures -- 3. Ultra fast chemical mismatch detection -- Labelling -- Solid phase -- Comments -- References -- 6. Mutation detection using T4 endonuclease VII -- 1. Introduction -- 2. The biology of Endo VII.

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