Cloning, Mapping and Sequencing Rodent Subtelomeric DNA

啮齿动物亚端粒 DNA 的克隆、定位和测序

• 批准号: 6915034
• 负责人: Harold RIETHMAN
• 金额: $ 26.9万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-25 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6915034
• 关键词: DNA; artificial chromosomes; gel electrophoresis; genetic library; genetic mapping; genome; laboratory mouse; laboratory rat; microarray technology; molecular cloning; nucleic acid sequence; plasmids; polymerase chain reaction; restriction endonucleases; telomere; DNA; artificial chromosomes; gel electrophoresis; genetic library; genetic mapping; genome; laboratory mouse; laboratory rat; microarray technology; molecular cloning; nucleic acid sequence; plasmids; polymerase chain reaction; restriction endonucleases; telomere

DESCRIPTION (provided by applicant): Cloning, mapping, and sequencing rodent subtelomeric DNA The goals of this project are to (1) clone large telomere-terminal DNA fragments from the mouse and rat genomes, (2) to collaboratively acquire finished DNA sequence extending from the euchromatic mouse and rat subtelomere regions to the terminal (TTAGGG) in tracts for individual telomeres, and (3) to validate the long range sequence organization predicted by these clone-based mapping and sequencing experiments by carrying out physical mapping experiments on cognate native chromosomal DNA. The DNA sequence organization of mammalian telomeres includes large stretches of highly similar duplicated and low-copy DNA adjacent to terminal telomere repeat sequences. This unusual sequence organization has led to significant complications with respect to mapping and sequencing subtelomeric DNA and connecting the physical endpoints of mammalian chromosomes with global clone-based physical maps and draft sequences. We propose to address this problem in rodent genomes using a specialized yeast artificial chromosome (half-YAC) system that permits propagation of large telomere-terminal DNA fragments as linear plasmids in yeast. Half-YACs from individual telomeres will be connected with global clone-based physical maps and draft sequences, then the particular repeat organization and DNA sequence of each subtelomeric region encompassed by the half-YACs will be deciphered without interference from duplicons derived from elsewhere in the genome. Physical mapping experiments on native chromosomal DNA using site-specific cleavage and pulsed-field gel electrophoresis methods will be carried out to validate the long-range sequence organization predicted by the clone-based mapping and sequencing experiments, and to detect potentially large polymorphism in specific subtelomere regions. This basic approach to completing telomere mapping and sequencing has worked successfully for most human telomeres; we propose here to apply it to mouse and rat telomeres.

描述（由申请人提供）： 克隆，映射和测序啮齿动物亚电体DNA 该项目的目标是（1）克隆大的端粒末端DNA片段，来自小鼠和大鼠基因组，（2），以协作获得成品的DNA序列，从位置鼠标和大鼠亚电位区域延伸到终端（TTAGGG）到终端（TTAGGG）在终端（TTAGGG）中，以通过这些范围的端粒和（3）序列的序列序列的序列（3）进行验证，并验证序列的序列，（3）序列的序列序列（3）对同源天然染色体DNA的物理映射实验。 哺乳动物端粒的DNA序列组织包括与末端端粒重复序列相邻的高度相似重复和低拷贝DNA的巨大拉伸。这个不寻常的序列组织导致了对映射和测序亚电体DNA的显着并发症，并将哺乳动物染色体的物理终点与基于全球克隆的物理图和草稿序列联系起来。我们建议使用专门的酵母人工染色体（Half-YAC）系统解决啮齿动物基因组中的这个问题，该系统允许将大型端粒末端DNA片段作为酵母中的线性质粒传播。来自单个端粒的半-YAC将与基于全球克隆的物理图和草稿序列相连，然后将涵盖半-YAC所包含的每个亚端粒区域的特定重复组织和DNA序列，而不会从基因组中其他位置得出的重复项的干扰。将使用位点特异性裂解和脉冲场凝胶电泳方法对天然染色体DNA进行物理映射实验，以验证通过基于克隆的映射和测序实验预测的远程序列组织，并检测特定特定的下层小区中潜在的大型多态性。这种完成端粒映射和测序的基本方法在大多数人类端粒中成功起作用。我们在这里建议将其应用于鼠标和大鼠端粒。