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.