HIGH RESOLUTION SEQUENCE READY MAPS

高分辨率序列就绪地图

• 批准号: 6344945
• 负责人: GLEN A EVANS
• 金额: $ 126.32万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-24 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344945
• 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：（根据申请人摘要改编）项目目标 1是产生高分辨率，序列准备，基于宇宙的地图， 大约30%的人类基因组，由11，20，21， 10、8、13、14和18。 从11号染色体开始， 将从低分辨率，基于YAC的地图开始， 正在建设中的几个实验室，包括圣地亚哥 基因组中心，并将进行YAC由YAC沿着最小平铺路径 低分辨率的地图。 拟议的方法将涉及 以下步骤获得高分辨率，基于宇宙的地图： 靶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， 用作杂交探针以鉴定YAC特异性的Cosplay in阵列 染色体特异性粘粒文库。 粘粒DNA 将通过碱裂解的自动化实施来制备 使用已经在圣地亚哥开发的机器人系统进行手术 基因组中心（Prepper，Ph.D.加纳博士开发的DNA制备机器人 和粘粒重叠群将在以下基础上组装： 限制性分析 将确定该酶的限制性图谱 通过间接末端标记/部分消化方法， 插入片段加上少量侧翼载体DNA 存在于载体中的位点，用酶如 EcoRI和寡核苷酸探针的凝胶转移杂交 特异于T3和T7载体序列，所述T3和T7载体序列存在于 切除NotI片段。 电泳和凝胶转移 杂交步骤将使用Labimap自动凝胶进行 在Genethon开发的分析系统。 重叠群的组装将是 基于重叠的限制性酶切图谱。 10至20倍的重叠群 将建造一个深度，并将覆盖每个YAC的至少90% 分身 这些地图中的空白，估计小于10 kb，间隔 平均每500 kb，将使用噬菌体，λ，P1 或从基因组文库分离的BAC克隆。 或者，如果间隙 如果小于10 kb，则它们可以通过直接从人类扩增来填充。 使用从侧翼重叠群末端产生的引物通过PCR扩增基因组DNA， 差距。 在用于高分辨率绘图之前，现有粘粒和YAC 文库将广泛地通过荧光原位表征 杂交，并对克隆进行了取样的详细分析 评估图书馆的质量和代表性。 在必要的情况下， 这些文库将补充额外的染色体特异性 本项目制作的图书馆，将质量和深度带入 所需范围。 埃文斯博士认为这一限速步骤 步骤为凝胶转移杂交。 使用4个Genethon 实验站和生产人员15人，持续的生产能力 预计每月将有40个青年活动中心。 预计5年期 在该项目中，这个吞吐量将外推到10亿字节对 完成30%的人类基因组图谱所需的比例。