Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.
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.
Narayanswami
S
S
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Lundgren
K
K
Hamkalo
B A
BA
D000818:Animals; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000648:ultrastructure*; D006048:Gold Colloid, Radioactive; D006801:Humans; D006824:Hybridization, Genetic; D008677:Metaphase; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D011817:Rabbits; D012091:Repetitive Sequences, Nucleic Acid; D014982:Xenopus laevis
D006048:Gold Colloid, Radioactive; D004247:DNA