IMPROVED TECHNOLOGIES FOR FULL LENGTH CDNA GENERATION

全长 CDNA 生成技术的改进

• 批准号: 6080531
• 负责人: JERRY PELLETIER
• 金额: $ 18.09万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6080531
• 关键词: DNA replication; Escherichia coli; RNA; animal tissue; bioengineering /biomedical engineering; complementary DNA; gene targeting; genetic library; introns; laboratory mouse; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; posttranscriptional RNA processing; technology /technique development; tissue /cell culture

The development of functional genomics resources is essential to understand and utilize information generated from genome sequencing projects. Central to the development of this technology is the creation of high quality cDNA resources and improved technologies for analyzing coding and noncoding mRNA sequences. In fact, the characterization of the full coding potential of a particular mRNA is the essential starting point when undertaking to investigate biological function. A full length cDNA allows one to predict transcription and translation initiation start sites, deduce certain protein characteristics based on primary amino acid sequence, predict transcription termination sites, and visually inspect the 5' and 3' untranslated regions for elements which may be involved in post- transcriptional regulation of gene expression. The analysis of several complete cDNAs for a given gene enables one to gather information on alternative splicing, alternative promoter usage, and alternative polyadenylation signals all events known to be important in regulating gene expression. The comparison of genomic and cDNA sequence is necessary for determining exon-intron structure and to document the occurrence of RNA editing - a post-transcriptional regulatory mechanism on which we have little information. In the current proposal, we aim to continue developing technologies for generating full-length cDNAs, and incorporate such technology into a process to yield high quality full-length cDNA libraries. Our specific aims are: i) To improve the efficiency and quality of first strand synthesis, ii) Establish a method that will determine whether a given set of cDNAs are full length, iii) Generate 5' selected cDNA libraries employing an improved affinity method, and iv) Implement an approach utilizing homologous recombination in E. coli by which the structure of cDNA clones can be tailored to individual needs. Together, these studies should yield higher quality cDNA libraries that can provide an important resource for novel gene discovery and provide reagents necessary for functional studies.

功能基因组学资源的开发对于理解和利用基因组测序项目产生的信息至关重要。 该技术开发的核心是创建高质量的cDNA资源和改进的技术，用于分析编码和非编码mRNA序列。 实际上，在研究生物学功能时，特定mRNA的完整编码潜力的表征是必不可少的起点。 全长的cDNA允许人们预测转录和翻译起始开始位点，根据原代氨基酸序列推断某些蛋白质特征，预测转录终止位点，并视觉检查5'和3'未翻译区域的5'和3'未翻译区域，以涉及可能参与基因表达后转录后调控的元素。 对给定基因的几个完整cDNA的分析使一个人能够收集有关替代剪接，替代启动子使用和替代聚腺苷酸化信号的信息，所有事件在调节基因表达中都很重要。 基因组和cDNA序列的比较对于确定外显子内结构并记录RNA编辑的发生是必要的 - 转录后调节机制，我们几乎没有信息。 在当前的建议中，我们旨在继续开发用于生成全长cDNA的技术，并将此类技术纳入生产高质量的全长cDNA库的过程中。 我们的具体目的是：i）提高第一链合成的效率和质量，ii）建立一种方法，该方法将确定一组cDNA是否全长，iii）产生5'选定的cDNA库，采用改进的亲和力方法，iv）实现一种利用E. Coli中的E. Coli中的cdna Clone的结构来缩小cdna clone的结构，从而实现一种方法。 这些研究共同产生更高质量的cDNA文库，可以为新型基因发现提供重要资源，并为功能研究提供必要的试剂。