In Situ Hybridization Protocols

About this book

Thirty-one detailed, practical chapters many by the originators themselves present easy-to-follow protocols of in situ hybridization techniques for mapping DNA sequences onto chromosomes, quantitation/localization of RNA in tissues, and detection of virus nucleic acids. The methods serve molecular biologists in most areas of basic and applied research, and in every sort of organism ranging from viruses to Drosophila to humans. The expert contributors provide step-by-step instructions for the preparation of probes for DNA, teleomeric repeats, cDNA sequences, small synthetic oligomers, whole cosmids, mega-size YACs, and chromosome-specific paints from somatic cell hybrids and flow-sorted chromosomes. They also present radioactive and nonradioactive probe labeling procedures with the use of nick translation, random priming, multicolor labeling, PRINS labeling of DNA and RNA, and PCR in situ hybridization. Color illustrations enhance the text. Troubleshooting tips, alternative ways of doing things, and informative explanations of why certain steps are done - aids not usually found in standard journal recipes - guarantee a significant difference in the outcome of experiments.

Contents

Preparation of Human Chromosomal Painting Probes from Somatic Cell Hybrids. Reverse Chromosome Painting. FISH Detection on DAPI Banded Chromosomes. Generation of Alphoid DNA Probes for Fluorescence In Situ Hybridization (FISH) Using the Polymerase Chain Reaction. In Situ Hybridization Using Synthetic Oligomers as Probes for Centromere and Telomere Repeats. Mapping Human YAC Clones by Fluorescence In Situ Hybridization using Alu-PCR from Single Yeast Colonies. FISH of Alu-PCR Amplified YAC Clones and Applications in Tumor Cytogenetics. Primed In Situ (PRINS) Labeling of DNA. Free Chromatin Mapping by FISH. Use of DNA-HALO Preparations for High-Resolution DNA In Situ Hybridization. In Situ Hybridization of Meiotic Prophase Chromosomes. Strand-Specific Fluorescence In Situ Hybridization for Determining Orientation and Direction of DNA Sequences. Radioactive In Situ Hybridization to Replication-banded Chromosomes. Gene Mapping Using 3H-labeled Heterologous Probes. Dual-Label Fluorescence In Situ Hybridization Detection of Individual Chromosomes in Sperm Using Ejaculated Semen. In Situ hybridization to Drosophila Polytene Chromosomes. In Situ Hybridization to Polytene Chromosomes of Drosophila Melanogaster and Other Dipteran Species. Microsurgical Isolation of Native Polytene Chromosomes of Drosophila Melanogaster for the In Situ Observation of Conformation and Distribution of Molecules. PCR In Situ Hybridization. Detection of Virus Nucleic Acids by Radioactive and Nonisotopic In Situ Hybridization. Detection of Viral Genomes. Primed In Situ (PRINS) Labeling of RNA. Hybridization Histochemistry Using Radiolabeled Oligodeoxyribonucleotide Probes. mRNA In Situ Hybridization to In Vitro Cultured Cells. Quantitative In Situ Hybridization Using Radioactive Probes in the Study of Gene Expression in Heterocellular Systems. Tissue Distribution of Gene Expression in Mammalian Development. Subcellular Location of mRNA by Electron Microscope Hybridization Histochemistry. Principle of Digital Imaging Microscopy. Application of In Situ Hybridization for the Detection of Virus Nucleic Acids. Positional Cloning and Multicolor In Situ Hybridization: Principles and Protocols. Characterization of Constitutive Marker Chromosomes in Humans. Index.

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