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 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.