附註:Includes bibliographical references and index.
Intro; Contents; List of contributors; Abbreviations; 1. Mapping protein/DNA interactions in vivo using ligation-mediated polymerase chain reaction; 1. Introduction; Methods used in determining protein/DNA interactions in vitro; Determining protein/DNA interactions in vivo; 2. The ligation-mediated polymerase chain reaction (LMPCR); Applications of LMPCR; 3. Mapping protein factor binding sites in vivo with LMPCR and DMS; DNA modification by DMS in vitro; DMS modification of DNA in vivo; Amplification of DMS/piperidine-cleaved DNA by LMPCR; Analysis of LMPCR reactions
4. Mapping nucleosomes using micrococcal nuclease and LMPCRIsolation of nuclei from cultured cells; Preparation of DNA from nuclei treated with MNase; Cleavage of genomic DNA with MNase; Acknowledgements; References; 2. Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos; 1. Introduction; 2. Outline of the method; 3. Formaldehyde cross-linking in staged Drosophila embryos; Preparation of fly cages and collection of staged embryos; Optimizing cross-linking conditions
4. Immunoprecipitation of cross-linked embryonic chromatin and PCR amplification of the immunoprecipitated DNA5. Analysing the enrichment of putative target sequences in the PCR-amplified DNA; Slot-blot analysis of the enrichment of putative PC target sequences; Mapping DNA target sites for Polycomb and GAGA factor in the Drosophila bithorax complex; 6. Concluding remarks; References; 3. Fission yeast chromosome analysis: fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (CHIP); 1. Introduction; 2. Fluorescence in situ hybridization (FISH) analysis of fission yeast
Preparation of probesCell fixation and cell-wall digestion; 3. Chromatin immunoprecipitation from fission yeast; Fixation of yeast cells to maintain protein localization; Preparation of chromatin extract; Immunoprecipitation of chromatin; Analysis of immunoprecipitated DNA sequences; Acknowledgements; References; 4. Isolation of vertebrate metaphase chromosomes and their analysis by FISH; 1. Introduction; 2. General equipment required for FISH; 3. Production of metaphase chromosomes as substrates for FISH; Production of fixed metaphase chromosome spreads
Production of long prometaphase chromosomesIsolation of suspensions of unfixed metaphase chromosomes; 4. Spreading fixed chromosomes; 5. Pretreatments of slides; Pretreatment of mitotic chromosome spreads; Salt extraction of isolated metaphase chromosomes; 6. Labelling DNA probes; Choice of label; Nick translation; Random priming; Labelling by PCR; Quantifying label incorporation; 7. Hybridization; Preparation of probes and slides; Hybridization; 8. Detecting hybridized probe; 9. Counterstaining and mounting; Simple counterstaining; Chromosome banding; Acknowledgements; References
摘要:The DNA of eukaryotes is packaged into chromosomes - each chromosome consisting of a very long molecule of DNA and various proteins (for example, histones), and the number of chromosomes being characteristic for the species concerned. Chromosome analysis can provide a great deal of information for many aspects of cellular genetics such as DNA replication, The book is structured in a methodical fashion - the introductory chapters are centred around analysis of chromatin with chapters on the mapping of protein: DNA interactions "in vivo" using ligation-mediated PCR and the mapping of chromatin-associated proteins by formaldehyde cross-linking. The next chapters concentrate on the study of whole chromosome structure, including: fission yeast chromosome analysis using FISH and CHIP, isolation of vertebrate metaphase chromosomes and their analysis by FISH, the study of vertebrate chromosome progression through mitosis, and the analysis of mammalian interphase chromosomes by immunofluorescence and FISH. There then follow chapters on FISH in whole-mount tissues and the analysis of the sub-structure of mammalian nuclei "in vitro."
