Jw. Breneman et al., THE DEVELOPMENT OF CHROMOSOME-SPECIFIC COMPOSITE DNA PROBES FOR THE MOUSE AND THEIR APPLICATION TO CHROMOSOME PAINTING, Chromosoma, 102(9), 1993, pp. 591-598
The speed and ease of human cytogenetic analysis has been greatly enha
nced by the technique of fluorescence in situ hybridization (FISH). No
n-radioactive fluorescently tagged complex DNA probes specific for ind
ividual chromosomes can be hybridized to conventionally obtained metap
hase chromosome spreads. Several chromosomes may be ''painted'' concur
rently by using combinations of different labeled probes. Surveys of c
hromosome breakage and rearrangement may be performed very quickly by
avoiding the time consuming process of GTG-banding. The application of
FISH to mouse cytogenetics would allow large scale molecular toxicolo
gy studies to be conducted on the effects of such environmental insult
s as potential carcinogens, mutagens and radiation. Progress has been
hampered, however, as the Mus musculus karyotype consists of 40 acroce
ntric chromosomes of approximately the same size, making the recogniti
on and separation of individual chromosomes very difficult. We now des
cribe the successful production and application of chromosome-specific
composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robe
rtsonian translocated chromosomes were isolated on a flow sorter and t
heir DNA subsequently amplified by degenerate oligonucleotide primer (
DOP) PCR. Small pools (300 copies) of each chromosome were denatured a
t 94-degrees-C then annealed with the primer at 30-degrees-C for 15 cy
cles. This was followed by 20 cycles at an annealing temperature of 62
-degrees-C. Additional amplification was performed at an annealing tem
perature of 62-degrees-C. The chromosome-specific DNA was labeled with
biotin 11-dUTP by nick translation and used for FISH. The usefulness
of the technique for translocation detection is demonstrated by analyz
ing chromosome exchanges induced in mice irradiated with Cs-132 gamma
rays.