Methods to analyze nuclear organizition of mammalian chromosomes in interphase nuclei

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Ordered arrangement of chromosomal DNAs into specific domains in nuclei has been proposed to fulfilll important roles in proper gene expression and DNA replication, as well as recombination especially in meiotic nuclei. Formation of non-B DNA structures such as triplex is thought important as a molecular mechanism determining spatial organization in nuclei. Triplex formation occurs readily in polypouine/polypyrimidine sequences leaving single-strand unpaired DNAs that can hybridize with other single-strand DNAs with significant complementarity. This will enable distantly spaced DNAs in genome sequences to associate with each other in interphase nuclei. Distantly spaced DNAs may also form transmolecular triplexes organizing themselves into an ordered array. To investigate mechanisms governing spatial organization of chromosomal DNAs in human and mouse nuclei, we develop multi-colors in situ binding assay methods for DNA probes with specific characteristics such as triplex formation, both in human mitotic and mouse meiotic nuclei. The method resembled fluorescence in situ hybridization used for detecting single-strand RNAs, thus was able to detect single-strand DNAs in nondenatured nuclei. Polyporine/polypyrimidine sequences, such as (GA/TC)n, (GAA/TTC)n and those present in human and mouse genomes, gave clear binding signals with different probe sequences resulting in different signal patterns. Using five pairs of filters optimized for excitation and emission lights that were controlled by a microcomputer, 5 colors of fluorescences were taken without mixing the colors with a cooled CCD camera.