HIGH RESOLUTION SEQUENCE READY MAPS

高分辨率序列就绪地图

• 批准号: 6241424
• 负责人: GLEN A EVANS
• 金额: $ 133.23万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-22 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6241424
• 关键词: artificial chromosomes; chromosome 21; chromosomes; fluorescent dye /probe; genetic library; genetic mapping; human genetic material tag; in situ hybridization; nucleic acid sequence; plasmids; pulsed field gel electrophoresis; restriction mapping; robotics

Description: (Adapted from the applicant's abstract) The goal of Project 1 is to produce high resolution, sequence-ready, cosmid-based maps for about 30 percent of human genome, consisting of chromosomes 11, 20, 21, 10, 8, 13, 14 and 18. Beginning with chromosome 11, the basic approach will start with the low-resolution, YAC-based maps that are presently under construction in several laboratories, including the San Diego Genome Center, and will proceed YAC-by-YAC along the minimum tiling path of the low-resolution maps. The proposed method will involve the following steps to obtain high-resolution, cosmid-based maps: A given target YAC will be separated from host chromosomes on a pulsed-field gel, extracted from the gel, and radiolabeled by random-hexamer priming for use as a hybridization probe to identify YAC-specific cosmids in arrayed chromosome-specific cosmid libraries by grid hybridization. Cosmid DNAs will be prepared by an automated implementation of the alkaline-lysis procedure using a robotic system already developed in the San Diego Genome Center (the Prepper, Ph.D. DNA prep robot developed by Dr. Garner at General Atomics) and cosmid contigs will be assembled on the basis of restriction analysis. Restriction maps of the cosmids will be determined by an indirect-end-label/partial-digestion method based on excision of the insert plus small amounts of flanking vector DNA by means of NotI sites present in the vector, partial digestion with an enzyme such as EcoRI, and gel-transfer hybridization with oligonucleotide probes specific to the T3 and T7 vector sequences present at each end of the excised NotI fragment. The electrophoresis and gel-transfer hybridization steps will be carried out using the Labimap automated gel analysis system developed at Genethon. Assembly of the contigs will be on the basis of overlapping restriction maps. Contigs from 10 to 20X in depth will be constructed and will cover at least 90 percent of each YAC clone. Gaps in these maps, estimated to be less than 10 kb and spaced on average every 500 kb, will be filled using bacteriophage, lambda, P1 or BAC clones isolated form genomic libraries. Alternatively, if gaps re less than 10 kb, they may be filled by direct amplification from human genomic DNA by PCR using primers produced from the contig ends flanking the gap. Before use for high-resolution mapping, existing cosmid and YAC libraries will be extensively characterized by fluorescence in situ hybridization, and detailed analysis of a sampling of clones carried out to assess quality and representation of the library. Where necessary, these libraries will be supplemented with additional chromosome-specific libraries produced by this project to bring quality and depth into the required range. Dr. Evans considers the rate-limiting step in this procedure to be the gel-transfer hybridization. Using 4 of the Genethon laboratory stations and a production staff of 15, a sustained throughput of 40 YACs per month is anticipated. Projected over the 5-year duration of the project, this throughput would extrapolate to the 1 gigabase pair scale required to complete maps for 30 percent of the human genome.

描述：（改编自申请人的摘要）项目的目标 1 是生成高分辨率、序列就绪、基于粘粒的图谱 约占人类基因组的 30%，由 11、20、21 号染色体组成， 10、8、13、14 和 18。从 11 号染色体开始，基本方法 将从目前基于 YAC 的低分辨率地图开始 包括圣地亚哥在内的多个实验室正在建设中 基因组中心，并将沿着最小平铺路径进行YAC-by-YAC 的低分辨率地图。 所提出的方法将涉及 按照以下步骤获得高分辨率、基于粘粒的图谱： 目标 YAC 将在脉冲场凝胶上与宿主染色体分离， 从凝胶中提取，并通过随机六聚体引发进行放射性标记 用作杂交探针来鉴定阵列中的 YAC 特异性粘粒 通过网格杂交获得染色体特异性粘粒文库。 粘粒DNA 将通过自动实施碱裂解来制备 使用圣地亚哥已经开发的机器人系统进行手术 基因组中心（Garner 博士开发的 Prepper、Ph.D. DNA 制备机器人 在通用原子公司）和粘粒重叠群将在此基础上组装 限制性分析。 将确定粘粒的限制性图谱 通过基于切除的间接末端标记/部分消化方法 通过 NotI 插入片段加上少量侧翼载体 DNA 载体中存在的位点，用酶部分消化，例如 EcoRI 和寡核苷酸探针的凝胶转移杂交 特定于 T3 和 T7 载体序列存在于每个末端 切除的NotI片段。 电泳和凝胶转移 杂交步骤将使用 Labimap 自动凝胶进行 Genethon 开发的分析系统。 重叠群的组装将是 基于重叠限制图谱。 重叠群 10 至 20X 将建造深度并将覆盖每个 YAC 的至少 90% 克隆。 这些地图中的间隙估计小于 10 kb 并且间隔 平均每 500 kb，将使用噬菌体、lambda、P1 进行填充 或从基因组文库分离的 BAC 克隆。 或者，如果有间隙 如果小于 10 kb，则可以通过人类直接扩增来填充它们 使用从重叠群末端侧翼产生的引物通过 PCR 检测基因组 DNA 差距。 在用于高分辨率作图之前，现有的 cosmid 和 YAC 文库将通过原位荧光进行广泛表征 杂交，并对克隆样品进行详细分析 评估图书馆的质量和代表性。 如有需要， 这些文库将补充额外的染色体特异性 该项目生成的库将质量和深度带入 所需范围。 埃文斯博士认为此过程中的速率限制步骤 程序为凝胶转移杂交。 使用 Genethon 的 4 个 实验室站和 15 名生产人员，持续的吞吐量 预计每月 40 个 YAC。 预计5年持续时间 在该项目中，该吞吐量将外推至 1 GB 对 完成 30% 人类基因组图谱所需的规模。