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© 1994 Oxford University Press

OTHER

Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization

Gabriele Senger+, Tania A.Jones, Helena Fldlerová2, Philippe Sanséau1, John Trowsdale1, Michael Duff3 and Denise Sheer*

1Human Cytogenetics Laboratory and 1Human Immunogenetics Laboratory, Imperial Cancer Research Fund 44 Lincoln's Inn Fields, London WC2A 3PX, UK 2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic Flemingovo Nám 2, 16637 Prague 6, Czech Republic 3Department of Physics and Astronomy, University College Gower Street, London WC1E 6BT, UK

*To whom correspondence should be addressed

Received March 28, 1994; Accepted May 26, 1994

Free DNA was prepared from routinely harvested and fixed cells for high resolution FISH mapping using either a sodium hydroxide/ethanol mixture or 70% formamide Hybridization signals from cosmid probes appeared as extended lines. The average length of signals on DNA prepared with sodium hydroxide was significantly greater than with formamide. A set of overlapping cosmlds from the HLA class II region was used to determine how precisely the actual overlap or gap between probes can be calculated from the measured overlap or gap between their signals. Lengths of the probe signals and their known kilobase lengths were used as an internal ruler. The mean values calculated from the measured length from 30 or more signals for each probe pair showed remarkable conformity with the known kilobase lengths of their overlaps and gaps. Immediately adjacent probes could also be ordered on the released DNA. These simple procedures dramatically Increase the speed with which relationships between probes can be determined during contlg construction.

+Present address: Institut fur Humangenetik und Anthropologie, Universität Jena, Kollegiengasse 10, 07740 Jena, Germany


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