CHROMATIN MAPPING BY FLUORESCENCE IN SITU HYBRIDIZATION

通过荧光原位杂交进行染色质定位

• 批准号: 3333320
• 负责人: BARBARA J. TRASK
• 金额: $ 24.33万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333320
• 关键词: DNA replication origin; biomedical equipment development; cell cycle; chromatin; color blindness; fluorescence; fluorescence microscopy; fragile X syndromes; gene dosage; gene expression; genetic mapping; genome; hamsters; human; human genetic material tag; in situ hybridization; nucleic acid sequence; sex chromosomes; sex linked trait; telomere

The long-term goals of this project are 1) to develop fluorescence in situ hybridization (FISH) as a tool for mapping and ordering the genome; 2) to understand the organization of sequences spaced 50 kbp to > 3 Mbp apart in chromatin during the cell cycle; and 3) to construct a long range map of Xq27-Xqter. The five specific aims of the proposed research are as follows: 1) To optimize one- and two-color detection of single copy sequences by FISH in terms of labeling efficiency, signal resolution, and image registration. 2) To compare the proximity of hybridization sites in chromatin of varying compaction. These targets include G1 interphase, prematurely condensed chromosomes, and (pro)metaphase chromosomes. The targets will be treated to extract proteins and decondense the chromatin. Emphasis will be on the selection of target(s) in which the relative spacing and order of sequences 50 kbp to 3 Mb apart can be most efficiently and accurately determined. 3) To describe the relationship between known linear distance and measured interphase and prometaphase distances for sequences separated by 50 kbp to 3 Mbp. This study will demonstrate whether FISH mapping is generally applicable to diverse chromosomal regions (telomeric vs. interstitial; G- dark vs. G-light banded). 4) To determine if the separation between sister chromatids at specific loci as cells progress from G1 to G2 can be used to derive additional map information. Sister chromatid separation will be studied in terms of the relative proximity of sequences to the telomere (human 4p16-pter) and to replication origins (DHFR region, Chinese hamster). 5) To use the FISH mapping techniques developed in aims 1-4 to construct a long range map of human chromosome Xq27-Xqter. FISH will be used a) to rapidly screen for Xq28 region-specific sequences from large-insert libraries; b) to confirm the relative spacing, orientation, and order of previously characterized PFG fragment clusters; c) to produce a fine map over regions of 1-2 Mbp by determining the spacing and order of region- specific sequences using FISH to interphase nuclei; and d) to determine if interphase distance measurements can be used to map disease loci by detecting disease-specific deletions.

该项目的长期目标是：1）开发原位荧光 杂交（FISH）作为基因组作图和排序的工具; 2） 了解间隔50 kbp至> 3 Mbp的序列的组织， 染色质在细胞周期;和3）构建一个长期的地图， XQ27-XQter。 拟议研究的五个具体目标如下： 1)为了优化单拷贝序列的单色和双色检测， FISH在标记效率、信号分辨率和图像方面的优势 登记 2)为了比较不同染色体组的染色质中杂交位点的接近程度， 压实 这些目标包括G1间期，过早浓缩 染色体和（前）中期染色体。 目标将被视为 来提取蛋白质和去浓缩染色质。 重点将放在 选择目标，其中序列的相对间距和顺序 50 kbp至3 Mb的间隔可以最有效和准确地确定。 3)描述已知直线距离与测量直线距离之间的关系 间隔50 kbp的序列的间期和前中期距离， 3Mbp。 这项研究将证明FISH定位是否普遍 适用于不同的染色体区域（端粒与间质; G- 暗对G-光带）。 4)为了确定姐妹染色单体之间的分离是否在特定的 随着细胞从G1进展到G2，基因座可用于推导额外的图谱 信息. 姐妹染色单体分离将根据 序列与端粒（人4p 16-pter）和端粒（人4p 16-pter）的相对接近度 复制起点（DHFR区，中国仓鼠）。 5)使用目标1-4中开发的FISH定位技术构建一个 人类染色体Xq 27-Xqter的长距离图谱。 FISH将用于a） 从大插入片段中快速筛选Xq 28区域特异性序列 B）确认相对间距、方向和顺序， 先前表征的PFG片段簇; c）产生精细图谱 在1-2 Mbp的区域上，通过确定区域的间隔和顺序， 使用FISH的特异性序列来间期核;和d）确定是否 间期距离测量可用于绘制疾病位点， 检测疾病特异性缺失。