FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

描述：（改编自申请人摘要）：长期目标 是开发新的流式细胞术（FCM）分析，利用多个，DNA 碱基特异性荧光染料和先进的FCM技术a）检测细微 染色质天然构型的变化， 生理刺激和B）提高个体的分辨率 染色体 这些研究的基本原理是基于这样一个概念， 染色质结构的调节是通过改变 DNA和核蛋白之间复杂的相互作用。 这些改变 改变DNA对碱基特异性荧光染料的可及性， 可被FCM有效检测。 结合的相关分析， 具有不同结合DNA模式的多种荧光染料可提供 这是一种评估染色质组织变化的独特方法。 这种新 技术为临床、结构和细胞提供了独特的优势， 生物学研究，包括，a）DNA荧光染料对细胞的应用 或染色体在相对无干扰的条件下， 保持接近天然构型，B）快速FCM分析， 将多种荧光染料结合中的相对变化与 细胞和c）需要少量细胞。 为了实现他们的长期目标，他们列出了以下具体的 目的：1）建立细胞DNA多荧光染色方法 和染色体，采用具有独特光谱的染料组合， 寿命和碱基对结合特性， 标记DNA上的特定区域，2）评估和优化 用于传统和独特FCM系统的分析技术， 将DNA-荧光染料探针相互作用与染色质中的调节相关联 结构，以及3）应用这种新的FCM技术分析 在选定的生物系统中染色质结构的渐进变化 以及用于表征单个鼠和人染色体。 他们 建议利用上一个赠款期间取得的进展 和专业知识，在我们的实验室进行细胞周期分析，染色质 结构、核蛋白生物化学、DNA损伤机制和 染色体 此外，他们将利用FCM系统， 洛斯阿拉莫斯国家流式细胞术资源，包括一个独特的流动 测量荧光寿命的系统结合常规的 FCM测量。 申请人声明，拟议研究将导致 新的细胞化学方法的发展， 以前不能通过流式细胞术获得结构和细胞功能 或其他分析系统。

DESCRIPTION: (Adapted from the Applicant's Abstract): The long term goal is to develop new flow cytometric (FCM) assays that utilize multiple, DNA base-specific fluorochromes and advanced FCM technology a) to detect subtle changes in the native configuration of chromatin in response to physiological stimuli and b) to improve resolution of individual chromosomes. The rationale for these studies is based on the concept that modulations in chromatin structure are brought about by alterations in the complex interaction between DNA and nucleoproteins. These alterations produce changes in DNA accessibility to base-specific fluorochromes that can be efficiently detected by FCM. Correlated analysis of the binding by multiple fluorochromes having different modes for binding to DNA can provide a unique approach for assessing changes in chromatin organization. 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. To achieve their long range goal they have listed the following specific aims: 1) to develop methods for multi-fluorochrome staining of DNA in cells and chromosomes, employing combinations of dyes that have unique spectral, lifetime and base-pair binding characteristics for sensitive and accurate labeling of particular regions on DNA, 2) to evaluate and optimize analytical techniques for use in conventional and unique FCM systems to correlate DNA-fluorochrome probe interactions with modulations in chromatin structure, and 3) to apply this new FCM technology for analysis of progressive changes in chromatin structure in selected biological systems and for characterization of individual murine and human chromosomes. They propose to take advantage of progress made during the previous grant period and expertise in our laboratory for cell-cycle analysis, chromatin structure, nucleoprotein biochemistry, DNA-damage mechanisms and chromosomes. Additionally, They will utilize FCM systems available through the National Flow Cytometry Resource at Los Alamos, including a unique flow system which measures fluorescence lifetime in conjunction with conventional FCM measurements. The applicant states the proposed studies will lead to the development of novel cytochemical approaches for correlating chromatin structure and cellular function not previously obtainable by flow cytometry or other analytical systems.