High-resolution DNA Fiber-FISH for genomic DNA mapping and colour bar-coding of large genes.
We have applied two-colour fluorescence in situ hybridization (FISH) to DNA fibers and combined it with digital imaging microscopy for the mapping of large cosmid contigs. The technique was validated using a set of unique plasmids and a cosmid contig both originating from the thyroglobulin (Tg) gene and previously mapped by restriction analysis. The resolution proved to be close to the theoretical lower limit of approximately 1 kb, ranging > or = 400 kb. Subsequently a 400 kb cosmid contig derived from a DMD-YAC was directly mapped by Fiber-FISH. The resulting map is in full agreement with the restriction map. Two-colour Fiber-FISH mapping thus showed to be capable for accurately sizing gaps and overlaps, and to identify chimeric or repeat sequence containing cosmids across a 400 kb region at once. The generated 400 kb 'colour bar-code' was subsequently used to map two DMD deletion breakpoints in patient DNA with an accuracy of 1-2 kb. The results underscore the value of this method for the delineation of chromosomal rearrangements for positional cloning and single patient clinical studies.
Florijn
R J
RJ
Department of Cytochemistry and Cytometry, State University Leiden, The Netherlands.
Bonden
L A
LA
Vrolijk
H
H
Wiegant
J
J
Vaandrager
J W
JW
Baas
F
F
den Dunnen
J T
JT
Tanke
H J
HJ
van Ommen
G J
GJ
Raap
A K
AK
D002678:Chimera / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D003116:Color; D003360:Cosmids / Q000235:genetics; D004247:DNA / Q000235:genetics*; D017353:Gene Deletion; D015321:Gene Rearrangement; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D009136:Muscular Dystrophies / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D013954:Thyroglobulin / Q000235:genetics
D004247:DNA; D013954:Thyroglobulin