High-resolution in situ hybridization using DNA halo preparations.
To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.
Wiegant
J
J
Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.
Kalle
W
W
Mullenders
L
L
Brookes
S
S
Hoovers
J M
JM
Dauwerse
J G
JG
van Ommen
G J
GJ
Raap
A K
AK
D002478:Cells, Cultured; D003360:Cosmids; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation
D004247:DNA