Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization.
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.
D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure*; D004247:DNA / Q000235:genetics*; D004254:DNA Nucleotidyltransferases; D003838:Deoxyadenine Nucleotides; D004330:Drosophila; D006046:Gold; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D011061:Poly A; D011071:Poly T
D003838:Deoxyadenine Nucleotides; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D006046:Gold; D004247:DNA; D004254:DNA Nucleotidyltransferases