MICRODISSECTED LIBRARIES OF HUMAN CHROMOSOMES

人类染色体显微切割文库

• 批准号: 2208458
• 负责人: DANIEL H JOHNSON
• 金额: $ 19.51万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208458
• 关键词: chromosomes; computer assisted sequence analysis; cytogenetics; dissection; gene duplication; genetic library; genetic mapping; genetic markers; genetic techniques; genome; human genetic material tag; in situ hybridization; molecular cloning; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; restriction mapping; sequence tagged sites; technology /technique development

At the present stage of the human genome project most chromosomes are poorly mapped. We propose to use chromosome-microdissection/PCR- amplification to isolate 10-20 megabase (Mb), region-specific DNA probe collections from human chromosome 1 in order to accelerate mapping. Although microdissected libraries have the potential to simplify marker assignment within a specific chromosomal region, this region is still quite large in molecular terms. Subsequent mapping within a region must still be performed by standard methods that are relatively slow. Therefore, we have developed a new strategy for mapping marker sequences from the microdissected libraries of DNA which take advantage of the fact that each microdissection library is in effect, a small human chromosome. The basic strategy is to hybridselect PCR amplimers from the microdissected libraries on sections of membrane Southern blots containing genomic DNA fractionated on CHEF gels. Each membrane slice contains genomic molecules in excess of 50 kb and thus any particular large molecule could be expected to specifically hybridize two or more PCR amplimers from an exclusive 10-20 Mb region of a chromosome ie. the linkage of microdissected amplimers is revealed by hybridization to the same, large DNA molecule on the membrane blot. By using multiple restriction endonucleases to cleave the CHEF-gel-fractionated-DNA it should be possible to build a relatively high resolution restriction map of the entire microdissected chromosomal region with STS markers derived from the sequence of the linked PCR amplimers. The long range goals of the work are to use and develop new technology, primarily based on chromosome dissection, which: (i) contributes to the construction of a 100 kb, STS marked physical map of human chromosomes 1; (ii) contributes to the isolation and analysis of specific chromosomal regions known to contain genes responsible for inherited or acquired human disease; (iii) contributes to a further understanding of heterochromatin structure and function and (iv) leads to the isolation of specific DNA probes that are useful in diagnosis of human diseases.

在人类基因组计划的现阶段， 地图很差。 我们建议使用染色体显微切割/PCR- 扩增以分离10-20兆碱基（Mb）区域特异性DNA探针 从人类1号染色体收集，以加速映射。 尽管显微切割的库有可能简化标记物 在特定的染色体区域内分配，该区域仍然是 从分子角度来说相当大。 区域内的后续映射必须 仍然可以通过相对较慢的标准方法来执行。 因此，我们开发了一种新的标记序列定位策略 从显微解剖的DNA文库中， 每个显微切割文库实际上是一个小的人类染色体。 基本策略是从PCR扩增物中杂交选择PCR扩增物。 膜Southern印迹切片上的显微切割文库 含有在CHEF凝胶上分级分离的基因组DNA。 每个膜片 含有超过50 kb的基因组分子，因此任何特定的 预计大分子可以特异性杂交两个或更多个 PCR扩增物从一个独特的10-20 Mb区域的染色体即。的 显微切割的扩增物的连接通过与 同样的大DNA分子在膜印迹上。 通过使用多个 限制性内切核酸酶切割CHEF-凝胶分级分离的DNA， 应该可以构建相对高分辨率的限制性图谱 整个显微切割的染色体区域与STS标记衍生 从连接的PCR扩增物的序列中。 的长期目标 这项工作是使用和开发新技术，主要是基于 染色体解剖，其中：（i）有助于构建一个 100 kb，STS标记的人类1号染色体物理图谱;（ii）有助于 涉及已知的特定染色体区域的分离和分析， 含有导致遗传或获得性人类疾病的基因;（iii） 有助于进一步了解异染色质结构， 功能和（iv）导致分离特异性DNA探针， 可用于人类疾病的诊断。