HIGH-RESOLUTION ORDERING OF DNA MARKERS BY MULTICOLOR FLUORESCENT IN-SITU HYBRIDIZATION OF PROPHASE CHROMOSOMES

To improve resolution for physical ordering of adjacent DNA loci, prop hase chromosomes were used for multi-color fluorescent in situ hybridi zation (FISH). The prophase chromosomes were prepared from cultured ly mphocytes by a thymidine synchronization, bromodeoxyuridine release te chnique and then treating the synchronized cultures with topoisomerase II inhibitors ICRF154 or ICRF193. Almost all mitotic figures exhibite d highly elongated prophase chromosomes without significant reduction of the mitotic index. Using multi-color FISH with these prophase chrom osomes, we were able to distinguish signals for loci separated by as l ittle as 50 kb, and determine their orientation. Furthermore, using th is prophase ordering system, we confirmed the linear order and defined the orientation of seven cosmid markers within a 360-kb region surrou nding D10S102, a locus that is closely linked to the disease locus in families segregating an allele causing multiple endocrine neoplasia II A (MEN2A). This prophase FISH system, by rapidly and precisely providi ng the linear order of loci that are very close, can expedite construc tion of fine cytogenetic maps and contribute to positional-cloning stu dies in which the precise ordering of DNA loci in a target region is c ritical.