Protocols for gene analysis / edited by Adrian J. Harwood.

Includes bibliographical references and index.

Basic recombinant DNA techniques -- Transformation of bacteria by electroporation -- Direct cloning of [lambda]g11 cDNA inserts into a plasmid vector -- PCR cloning using T-vectors -- Thermal cycle dideoxy DNA sequencing -- In vitro mutagenesis -- Ordered deletions using exonuclease III -- Site-directed mutagenesis using a double-stranded DNA template -- Site-directed mutagenesis using a uracil-containing phagemid template -- Construction of linker-scanning mutations by oligonucleotide ligation -- Construction of linker scanning mutations using the polymerase chain reaction -- Localized random polymerase chain reaction mutagenesis -- Genomic structure -- Simultaneous isolation of RNA and DNA from tissues and cultured cells -- Physical mapping of the human genome by pulsed field gel electrophoresis -- Field inversion gel electrophoresis -- Enhanced chemiluminescent detection of horseradish peroxidase labeled probes -- Nonradioactive oligonucleotide probe labeling -- Analysis of DNA restriction enzyme digests by two-dimensional electrophoresis in agarose gels -- Inverse polymerase chain reaction -- Sequence variations -- Use of silver staining to detect nucleic acids -- Nonradioactive method for the detection of single-strand conformational polymorphisms (SSCP) -- Temperature gradient gel electrophoresis (TGGE) for the detection of polymorphic DNA and RNA -- TGGE in quantitative PCR of DNA and RNA -- PGK-PCR clonality assay (PPCA) -- Direct sequencing of PCR products -- Gene expression -- Use of riboprobes for the analysis of gene expression -- Quantification of absolute amounts of cellular messenger RNA by RNA-excess solution hybridization -- Measurements of rate of transcription in isolated nuclei by nuclear run-off assay -- RNA polymerase II in vitro transcription system -- S1 mapping using single-stranded DNA probes -- Single primer-mediated polymerase chain reaction -- Protein-DNA interactions -- In vivo DNA footprinting by linear amplification -- DNA photofootprinting with Rh(phi)₂bpy³ -- Gel retardation assay -- Southwestern assay -- Cloning DNA binding proteins from cDNA expression libraries using oligonucleotide binding site probes -- Protein function -- 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification -- Production of ³⁵S-labeled proteins in E. coli and their use as molecular probes -- Preparation and ligand screening of a [lambda]g11 lysogen library.

This volume provides a set of protocols for the analysis of cloned genes. A set of techniques is described for the basic manipulation and mutagenesis of the cloned sequences. The rest of the volume is organized into a collection of techniques appropriate to the analytical route to be investigated; these include genome structure, sequence variation, gene expression, and protein function. Finally, a number of methods are described to take the step from the first gene to those with which it interacts.

English.