The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting.
The speed and ease of human cytogenetic analysis has been greatly enhanced by the technique of fluorescence in situ hybridization (FISH). Non-radioactive fluorescently tagged complex DNA probes specific for individual chromosomes can be hybridized to conventionally obtained metaphase chromosome spreads. Several chromosomes may be "painted" concurrently by using combinations of different labeled probes. Surveys of chromosome 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 toxicology studies to be conducted on the effects of such environmental insults as potential carcinogens, mutagens and radiation. Progress has been hampered, however, as the Mus musculus karyotype consists of 40 acrocentric chromosomes of approximately the same size, making the recognition and separation of individual chromosomes very difficult. We now describe the successful production and application of chromosome-specific composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robertsonian translocated chromosomes were isolated on a flow sorter and their DNA subsequently amplified by degenerate oligonucleotide primer (DOP) PCR. Small pools (300 copies) of each chromosome were denatured at 94 degrees C then annealed with the primer at 30 degrees C for 15 cycles. This was followed by 20 cycles at an annealing temperature of 62 degrees C. Additional amplification was performed at an annealing temperature 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 analyzing chromosome exchanges induced in mice irradiated with 137Cs gamma rays.
Breneman
J W
JW
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-9900.
Ramsey
M J
MJ
Lee
D A
DA
Eveleth
G G
GG
Minkler
J L
JL
Tucker
J D
JD
D000818:Animals; D001483:Base Sequence; D006690:Bisbenzimidazole; D002478:Cells, Cultured; D014128:Chromomycin A3; D002875:Chromosomes; D004247:DNA / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D015342:DNA Probes; D005260:Female; D005434:Flow Cytometry; D005720:Gamma Rays; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007621:Karyotyping; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D014178:Translocation, Genetic
D015342:DNA Probes; D004247:DNA; D014128:Chromomycin A3; D006690:Bisbenzimidazole