Faculty Research 1980 - 1989

Title

Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.

Document Type

Article

Publication Date

1989

Keywords

Cells-Cultured, Chromosome-Mapping, Chromosomes: ul, DNA: ul, Gold-Colloid-Radioactive, Human, Hybridization, Metaphase, Mice, Microscopy-Electron: mt, Rabbits, Repetitive-Sequences-Nucleic-Acid, SUPPORT-U-S-GOVT-P-H-S, Xenopus-laevis

JAX Source

Scanning Microsc Suppl 1989;3:65-76

Grant

GM23241, GM07311

Abstract

Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification.

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