EXON DETECTION IN VERTEBRATE DNA

脊椎动物 DNA 中的外显子检测

• 批准号: 2208648
• 负责人: SUSAN M BERGET
• 金额: $ 20.05万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2208648
• 关键词: DNA; artificial chromosomes; chromosomes; genetic library; genetic manipulation; genetic techniques; genetic transcription; genome; human genetic material tag; method development; molecular genetics; oligonucleotides; plasmids; polymerase chain reaction; sex chromosomes; structural genes; tissue /cell culture; transfection /expression vector

The long term goal of this project is to develop a method to identify rapidly individual transcription units in the human genome using a 3'- terminal exon trapping strategy developed during the first period of this award. This strategy utilizes a mammalian expression vector containing an incomplete transcription unit lacking a 3'-terminal exon. Only in the presence of inserted genomic DNA does the vector produce stable poly(A)+RNA upon transfection of cultured cells. RNA, and thereby the gene from which it originated, is detected and obtained by RT/PCR amplification. In the next five years we plan to: 1) Refine the technique. We will explore technique modifications designed to minimize false positives an maximize sensitivity. We will also develop an in vitro trapping technique. 2) Extend trapping to more complex sources of DNA. We have just trapped our first exons rom cosmids. We wish to extend this analysis, with an emphasis on determining technique efficiency when applied to cosmid and YAC DNA. 3) Identify potential trnscription units within a specified chromosomal region. To test the efficiency and sensitivity of our exon trapping protocol we plan to identify transcriptional units within the p22 and p23 regions of human chromosome 6. 4) Creation of X chromosome specific libraries. Using an arrayed library of X-specific cosmids and YACs presently available in the Baylor Genome Center as sources of genomic DNA, we plan to isolate X chromosome 3'- terminal exons. Isolated exons will be sequenced, catalogued, and arrayed for use by other investigators.

这个项目的长期目标是开发一种方法来识别 使用3 '-脱氧核糖核酸快速分离人类基因组中的转录单位。 末端外显子捕获策略是在本研究的第一阶段开发的， 奖 该策略利用了含有一种哺乳动物表达载体， 一个不完整的转录单位，缺少3 '端外显子。 只有在 插入的基因组DNA的存在下，载体是否产生稳定的poly（A）+RNA 转染培养的细胞后。 RNA，因此， 其来源、检测和获得通过RT/PCR扩增。 在 未来五年，我们计划： 1)完善技术。 我们将探索技术改进， 以最小化假阳性并最大化灵敏度。 我们还将开发 一种体外诱捕技术 2)将捕获扩展到更复杂的DNA来源。 我们刚刚困住了 我们的第一个外显子来自Cosmos。 我们希望扩展这种分析， 当应用于粘粒和YAC时，强调确定技术效率 DNA. 3)识别特定染色体内的潜在转录单位 地区 为了测试我们的外显子捕获的效率和灵敏度 我们计划在p22和p23中识别转录单位 人类6号染色体的区域。 4)X染色体特异性文库的创建。 使用阵列库 目前在Baylor基因组中可获得的X特异性cosplay和YAC 作为基因组DNA的来源，我们计划分离X染色体3 '- 末端外显子 分离的外显子将被测序，分类，排列 供其他研究人员使用。