CORE--MOLECULAR BIOLOGY DNA SEQUENCING CORE

建议在#年建立DNA测序核心设施。 使用与俄亥俄州东北部多用途有关的调查人员 关节炎中心。尽管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范围相当广泛 而且是特定于项目的，大多数实验室都将受益于核心 专门用于DNA序列测定的设备。这样做的理由是 1)DNA测序在任何分子中的某个点都是必需的 以生物学为导向的实验方法，以及2)它并不总是昂贵的 有效地为每个实验室建立单独的方案，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，但不需要具备以下方面的特定知识 更大的项目。4)现场测序设施可以极大地 提高关节炎中心研究的整体生产力 实验室通过更高效、更高效地执行这项任务 精确度超过了个人可能达到的水平。5)核心 设施将为调查人员腾出时间从事更具创造性的工作 追求。 该核心的主要目标将是协助这些调查人员 已经参与了分子生物学方案。为了成为 实用，它不会服务于执行和分子生物学技术 而不是DNA序列分析。调查人员将负责 在提交前准备样品进行测序。 样品制备方案将由测序核心提供 为了确保核心设施得到有效利用 并确保成功。一般来说，调查人员将负责 1)将DNA片段连接到多联体(克隆位点)中 DNA常用的测序载体(如M13或Bluescrip) 用Sanger双脱氧核苷酸方法进行序列分析。2)在这种情况下 在M13中，研究人员将分离出含有插入DNA和 制备单链模板DNA。3)在质粒载体的情况下 研究人员可以使用一种 辅助噬菌体，或制备适合测序的双链质粒。 然后，核心设施将负责执行测序 反应、运行测序凝胶、生成放射自显影、读取 DNA序列，并将序列记录为文件，可用于 调查员进一步分析。 核心的第二个目标将是作为一个存储库 患有骨性关节炎的家庭成员的基因组DNA显示家族性 遗传，以及来自骨性关节炎疾病亚群的患者。从这些DNA中提取 个人将可供其他调查人员进行协作 基础。此外，核心还将对这个DNA库进行筛选 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.