CORE--MOLECULAR BIOLOGY DNA SEQUENCING CORE

建议建立一个DNA测序的核心设施， 使用与东北部俄亥俄州多用途 关节炎中心。 虽然分子生物学程序的范围 与关节炎中心相关的实验室使用的方法相当广泛 并且是特定于项目的，大多数实验室将受益于一个核心 专门用于DNA序列测定的设施。 这样做的理由 1）DNA测序在任何分子生物学中的某个时刻都是必需的， 生物学为导向的实验方法，2）它并不总是成本 每个实验室建立单独的协议，购买 装备和培训必要的人员。 3)DNA序列分析 是劳动和时间密集型的，但不需要特定的知识， 更大的项目 4)现场测序设备可以大大 提高关节炎中心研究的整体生产力 通过更有效地执行这项任务， 精确度高于个体。 5)核心 设施将腾出时间，让调查人员从事更有创造性的 追求 核心小组的主要目标是协助这些调查员 已经参与了分子生物学实验。 为了成为 实际上，它不会用于执行和分子生物学技术， 除了DNA序列分析 调查人员将负责 在提交前准备测序样品。 测序中心将提供样品制备方案 为了确保核心设施得到有效利用， 并确保成功。 一般来说，研究人员将负责 1)将DNA片段连接到多接头（克隆位点）中， 通常用于DNA测序的测序载体（即M13或Bluescript） 通过桑格双脱氧核苷酸法进行序列分析。 2)的情况下 研究人员将分离含有插入DNA的M13克隆， 制备单链模板DNA。 3)在质粒载体的情况下 研究者可以使用 辅助噬菌体，或制备适于测序双链质粒。 然后，核心设施将负责进行测序 反应，运行测序凝胶，生成放射自显影图，阅读 DNA序列，并将该序列记录为可用于 研究人员的进一步分析。 该核心的第二个目标是作为一个储存库， 来自患有OA的家庭成员的基因组DNA显示家族性 遗传，并从患者的OA疾病子集。 DNA从这些 个人将提供给其他调查人员的合作， 基础 此外，核心将发挥作用，筛选这个DNA库， II型胶原基因突变。

It is proposed that a DNA sequencing core facility be established fore the use of investigators associated with the Northeast Ohio Multipurpose Arthritis Center. Although the scope of molecular biology procedures used by laboratories associated with the Arthritis Center is quite broad and are project- specific, most laboratories would benefit from a core facility dedicated to DNA sequence determination. The rationale for this is that 1) DNA sequencing is required at some point in any molecular biology oriented experimental approach, and 2) it is not always cost effective for each laboratory to set up individual protocols, purchase equipment and train the necessary personnel. 3) DNA sequence analysis is labor and time intensive, but does not require specific knowledge of the larger project. 4) An on site sequencing facility could greatly enhance the overall productivity of Arthritis Center research laboratories by performing this task more efficiently and with greater precision than would be possible on an individual basis. 5) The core facility would free time for investigators to engage in more creative pursuits. The primary objective of the core would be to assist those investigators already engaged in molecular biology protocols. In order to be practical, it would not serve to perform and molecular biology techniques other than DNA sequence analysis. Investigators would be responsible for preparation of samples for sequencing in advance of submission. Protocols for sample preparation will be provided by the sequencing core in order to assure that the core facility will be efficiently utilized and to assure success. In general, investigators will be responsible for 1) ligation of DNA fragments into the polylinker (cloning site) of sequencing vectors commonly in use (i.e. M13 or Bluescript) for DNA sequence analysis by the Sanger dideoxynucleotide method. 2) In the case of M13, investigators will isolate M13 clones containing insert DNA and prepare single stranded template DNA. 3) In the case of plasmid vectors the investigator may either prepare single stranded template using a helper phage, or prepare double stranded plasmid suitable for sequencing. The core facility will then be responsible for performing the sequencing reactions, running sequencing gels, generating autoradiograms, reading DNA sequence and recording the sequence as a file that can be used for further analysis by the investigator. The second objective of the core would be to serve as a repository for genomic DNA from members of families afflicted with OA showing familial inheritance, and from patients with OA disease subsets. DNA from these individuals would be available to other investigators on a collaborative basis. Additionally, the core will function to screen this DNA bank for mutations in the type II collagen gene.