FLOW CYTOMETRIC ANALYSIS OF MULTIPLE DNA FLUOROCHROMES

描述：(改编自申请者摘要)：长期目标 是开发利用多个DNA的新的流式细胞仪(FCM)分析 碱基特定的荧光染料和先进的FCM技术a)检测细微的 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。这些研究的理论基础是基于这样一个概念 染色质结构的调制是由 DNA和核蛋白之间的复杂相互作用。这些改动 使DNA对碱基特异的荧光染料的可及性发生变化，从而 被FCM有效检测到。结合的相关性分析 具有与DNA结合的不同模式的多个荧光染料可以提供 一种评估染色质组织变化的独特方法。如此新奇 技术为临床、结构和细胞提供了明显的优势 生物学研究，包括：a)DNA荧光染料在细胞中的应用 或染色体在相对不受干扰的条件下，因此染色质 保持接近本机配置，b)快速FCM分析 多个荧光素结合的相对变化与 细胞和c)对细胞数量的要求。 为了实现他们的长期目标，他们列出了以下具体内容 目的：1)建立细胞内DNA的多重荧光染色方法 和染色体，使用具有独特光谱的染料组合， 寿命和碱基对结合特征，以实现灵敏和准确 DNA上特定区域的标记，2)评估和优化 用于常规和独特的FCM系统的分析技术 DNA-荧光染料探针相互作用与染色质调控的关系 结构，以及3)将这种新的FCM技术应用于分析 部分生物系统中染色质结构的渐进性变化 以及对单个小鼠和人类染色体的鉴定。他们 建议利用前一批款期取得的进展 和我们实验室在细胞周期分析、染色质方面的专业知识 结构，核蛋白生物化学，DNA损伤机制和 染色体。此外，他们将利用通过以下途径提供的FCM系统 洛斯阿拉莫斯的国家流式细胞术资源，包括一种独特的Flow 一种结合常规方法测量荧光寿命的系统 FCM测量。申请者表示，拟议的研究将导致 染色质相互关联的细胞化学新方法的发展 以前无法通过流式细胞术获得的结构和细胞功能 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.