GENERATION OF CHROMOSOME REGION-SPECIFIC CDNA LIBRARIES

染色体区域特异性 CDNA 文库的生成

• 批准号: 2252923
• 负责人: PAUL D SIEBERT
• 金额: $ 27.29万
• 依托单位: CLONTECH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-15 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2252923
• 关键词: biotechnology; cell cycle; chromosomes; complementary DNA; genetic library; genetic mapping; genome; human genetic material tag; human tissue; in situ hybridization

This proposal describes the application of a new technology, termed "preparative in-situ hybridizational (Prep.-I.S.H.) for generating cDNA libraries that map to defined legions of the human genome Prep.-I.S.H. is a combination of two established technologies, in-situ hybridization to metaphase chromosomes, and chromosome microdissection. Briefly, the procedure calls for hybridization of an uncloned cDNA library on the ends with primer binding sites, to metaphase chromosome spreads. The cytogenetically defined legion of interest is then microdissected, and the cDNAs that are bound to the dissected legion are then amplified using the PCR. By specifying the initial source of the cDNA probe library the method can be tailored so that tissue-specific, developmental-stage specific and chromosome legion-specific cDNAs can be generated. This technology will increase the speed of attaining the goals of the NCHGR by allowing certain regions of the genome to be targeted, as well as providing a method for isolating coding regions without the implication of inter and intra-genic DNA sequences. We propose to apply the Prep.-I.S.H. technology, when optimized, to generate legion-specific human chromosome cDNA libraries. The commercialization of such libraries will provide a valuable user friendly and cost effective resource for human genome studies.

这项提案描述了一项名为 “制备性原位杂交法(Prep.-I.S.H.)” 映射到人类基因组的特定军团的文库-I.S.H.是 两种成熟技术的组合，原位杂交 中期染色体和染色体显微切割。简而言之， 该程序要求在末端杂交未克隆的cdna文库。 有了引物结合部位，才能使中期染色体扩散。这个 然后对细胞遗传学定义的感兴趣军团进行显微解剖，然后 与解剖军团结合的cDNA然后使用 聚合酶链式反应。通过指定cDNA探针库的初始来源，该方法 可以定制为特定于组织、特定于发育阶段和 可以产生染色体军团特有的cDNA。这项技术将 通过允许某些项目加快实现NCHGR目标的速度 作为目标的基因组区域，以及提供一种 分离不含基因间和基因内的编码区 DNA序列。我们建议在以下情况下应用Prep.-I.S.H.技术 优化，以产生军团特异的人类染色体cDNA文库。 这些图书馆的商业化将提供一个有价值的用户 友好且经济实惠的人类基因组研究资源。