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Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization

Wu, Madeline and Davidson, Norman (1981) Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization. Proceedings of the National Academy of Sciences of the United States of America, 78 (11). pp. 7059-7063. ISSN 0027-8424.

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A transmission electron microscope method for gene mapping by in situ hybridization to Drosophila polytene chromosomes has been developed. As electron-opaque labels, we use colloidal gold spheres having a diameter of 25 nm. The spheres are coated with a layer of protein to which Escherichia coli single-stranded DNA is photochemically crosslinked. Poly(dT) tails are added to the 3' OH ends of these DNA strands, and poly(dA) tails are added to the 3' OH ends of a fragmented cloned Drosophila DNA. These probe-dA strands are hybridized in situ to polytene chromosome squashes. Gold spheres are linked to the hybridized probe-dA strands by A· T base pairing. The sphere positions relative to the chromosome bands can be observed by transmission electron microscopy. The method shows low background and high resolution.

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Additional Information:© 1981 by the National Academy of Sciences. Contributed by Norman Davidson, August 3, 1981. We are indebted to Dr. Welcome Bender for the gift of cDm2848 and cDm2188 DNAs and for much advice and discussion, to Dr. Tony Weil for the pC1T19 DNA, and to Dr. Herschel Mitchell for advice about chromosome preparations. This research was supported by National Institutes of Health Grant GM 20927. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:colloidal gold spheres; A•T base pairing; photochemical crosslinking
Issue or Number:11
Record Number:CaltechAUTHORS:WUMpnas81
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10508
Deposited By: Tony Diaz
Deposited On:14 May 2008
Last Modified:03 Oct 2019 00:10

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