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Simultaneous Visualization of Chromosome Bands and Hybridization Signal Using Colloidal-Gold Labeling in Electron Microscopy
Raouf Fetni, Regen Drouin, Nicole Lemieux, Paul-Emil Messier and Claude-Lise Richer
Proceedings of the National Academy of Sciences of the United States of America
Vol. 88, No. 23 (Dec. 1, 1991), pp. 10916-10920
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2358364
Page Count: 5
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Electron microscopy (EM) is seldom used with in situ hybridization to localize DNA sequences because banding methods for chromosome identification could not be coupled to EM techniques. We have applied an immunochemical replication-banding method specific for EM to solve this problem. A thymidine synchronization/BrdUrd release protocol allows BrdUrd incorporation only into late replicating bands. A biotinylated DNA probe is hybridized in situ to its complementary sequence. The biotinylated probe and the BrdUrd-substituted DNA are simultaneously localized by different reporter/detection systems using different-sized colloidal gold particles as electron-dense tags. We demonstrate the high precision of this mapping procedure by localizing on long prophase chromosomes (>1000 bands per haploid set) the pXBR-1 sequence to a small subregion of the centromeric subband Xp11.1-Xq11.1. This localization to a part of an individual prophase subband is the most precise localization ever reported on human banded mitotic chromosomes.
Proceedings of the National Academy of Sciences of the United States of America © 1991 National Academy of Sciences