DEVELOPMENT OF SYSTEMS FOR RAPID CHROMOSOME MAPPING

快速染色体作图系统的开发

• 批准号: 3500484
• 负责人: PAUL D SIEBERT
• 金额: $ 5万
• 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: 1992-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3500484
• 关键词: DNA; chromosomes; complementary DNA; genetic library; genetic mapping; high performance liquid chromatography; human genetic material tag; method development; molecular biology; molecular cloning; nucleic acid chemical synthesis; nucleic acid hybridization; nucleic acid probes; oncogenes; plasmids; polymerase chain reaction; synthetic nucleic acid; transfection; transfection /expression vector

We propose to develop simple and versatile systems to map mammalian genes to individual chromosome and subchromosome bands by application of the polymerase chain reaction (PCR) technique. Amplification is performed by utilizing a universal PCR template-adaptor and primer (cassette) which is inserted randomly into the chromosomal DNA by ligation to combinations of restriction enzyme digests. A permanent collection of the amplified chromosomal DNA will then be established by cloning into a plasmid vector. With this system chromosome mapping can then be performed in several ways; (1) By hybridization of cDNA or gene fragment probes to amplified chromosomal DNA immobilized on hybridization membranes, (2) By PCR analysis to collections of the cloned chromosomal DNA with gene specific primer sets, and (3) By use of the cloned DNA as chromosome specific hybridization probes. In phase I we will evaluate the feasibility of this approach using PCR amplified whole gemonic DNA. This will be followed by amplification of whole metaphase cell chromosome dissections to optimize the manipulation and processing of chromosomes. Phase II will extend the methodology to dissected individual chromosomes and construct libraries from all 23 human chromosomes, as well as subchromosomal bands in regions of particular interest.

我们建议开发简单而通用的系统来映射哺乳动物 基因到单个染色体和亚染色体带的应用， 聚合酶链反应（PCR）技术。 扩增是 通过使用通用PCR模板-衔接子和引物进行 （盒），其通过以下方式随机插入染色体DNA中： 连接至限制性内切酶的组合。 永久 然后通过以下步骤建立扩增的染色体DNA的收集： 克隆到质粒载体中。 利用该系统， 然后以几种方式进行：（1）通过cDNA或基因的杂交 片段探针扩增染色体DNA固定在 杂交膜，（2）通过PCR分析收集的 用基因特异性引物组克隆染色体DNA，和（3）通过使用 克隆的DNA作为染色体特异性杂交探针。 一期 我们将使用PCR扩增来评估这种方法的可行性 完整的基因组DNA 这将是随后的放大整体 中期细胞染色体解剖以优化操作， 处理染色体。 第二阶段将把方法扩大到 解剖单个染色体，并从所有23条染色体中构建文库 人类染色体，以及亚染色体带的区域， 特别的兴趣。