EXON DETECTION IN VERTEBRATE DNA

这个项目的长期目标是开发一种方法来识别 在人类基因组中使用3‘- 末端外显子捕获策略是在本研究的第一阶段发展起来的 获奖。这种策略利用了一种哺乳动物表达载体 不完整的转录单位，缺少3‘端外显子。仅限于在 插入的基因组DNA是否能产生稳定的Poly(A)+RNA 在对培养的细胞进行转染时。RNA，以及由此产生的基因 它的来源、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。我们将探索设计的技术改进 将假阳性降至最低，并最大限度地提高灵敏度.我们还将发展 一种体外诱捕技术。 2)将捕获范围扩大到更复杂的DNA来源。我们刚刚被困住了 我们的第一个外显子来自宇宙。我们希望通过一个 重视确定COSMID和YAC的技术效率 DNA 3)确定特定染色体内的潜在转录单位 区域。来测试我们外显子捕获的效率和敏感性 我们计划识别p22和p23中的转录单位 人类6号染色体的区域。 4)X染色体特异性文库的构建。使用阵列库 目前在贝勒基因组中可获得的X特定的宇宙染色体和YAC 中心作为基因组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、编目和排列 供其他调查人员使用。

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.