FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

流式细胞术（FCM）分析dna结合差异

Flow cytometric (FCM) analyses of the differential in DNA-binding by multiple base-specific fluorochromes can provide a unique approach for detecting metabolic changes in the native state of chromatin in individual cells responding to changes in the physiological state of the population. This rationale is based on the premise that subtle rearrangements in chromatin organization involve modulations in DNA- nucleoprotein interactions resulting in disproportionate changes in DNA accessibility to base-specific fluorochromes that can be efficiently detected by FCM. Such new technology provides distinct advantages for clinical, structural, and cell biology studies including, (a) the application of DNA fluorochromes to cells or chromosomes under relatively non-perturbing conditions so that chromatin remains close to the native configuration, (b) rapid FCM analyses for correlating the relative changes in binding by multiple fluorochromes in cells and (c) requirement of small numbers of cells. The long term goal is to develop this new FCM approach to increase the sensitivity for detecting cell cycle-related changes in chromatin organization and for identifying individual chromosomes. Achieving this goal will require (1) detailed spectral studies on selected, DNA based-specific fluorochromes bound to DNA and chromatin, (2) utilizing unique FCM spectroscopic and conventional instrumentation to quantitate relative changes in binding features of multiple DNA fluorochromes to chromatin in cells and chromosomes, (3) applying new computer analyses methods that can better correlate binding by multiple fluorochromes. Specific Aim 1 is perform spectrofluorometric studies on DNA-specific dyes, including new, ultrasensitive fluorochromes, such as TOTO and YOYO, to determine fluorescence lifetime, energy transfer, and other dye-dye interactions that will provide the rationale for developing multifluorochrome labeling techniques. Specific Aim 2 is to evaluate the sensitivity of unique analytical approaches utilizing recently developed FCM instrumentation, including our phase sensitive detection FCM, the Fourier transform FCM, as well as our multiparameter three laser FCM system and the two laser, chromosome analysis FCM. Specific Aim 3 is to apply new computer analysis methods, including the IDYLK analysis programs, cluster analysis, differential fluorescence and ratio analyses as developed in this laboratory to correlate differential changes in DNA accessibility to base-specific fluorochromes with the cell cycle-related modifications of nucleoproteins to obtain a better understanding of the molecular basis for the alterations in the cytochemical patterns of DNA accessibility.