STREAMLINING DIFFERENTIAL DISPLAY TECHNOLOGY

简化差异显示技术

• 批准号: 2896332
• 负责人: PENG LIANG
• 金额: $ 24.27万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-14 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2896332
• 关键词: DNA primers; biomedical automation; biomedical equipment development; cytogenetics; gene expression; genetic techniques; in situ hybridization; method development; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; robotics

DESCRIPTION: (Applicant's Description) Of the estimated 100,000 individual genes encoded by the human genome, approximately 10-20% are expressed by the average cell. The expression of this subset of genes is a major determinant of a cell's properties. In addition to the control of cellular phenotype and the normal physiological processes of an cancer. Therefore understanding of the mechanisms of these normal and pathological processes requires organism. alterations in gene expression also underlies the etiology of diverse pathological processes such as identification. isolation and characterization of differentially expressed genes. Differential display (DD) technology was developed recently for this purpose and has emerged as the most commonly used method for the identification and cloning of differentially expressed genes. Despite of its wide use, the power of DD has not yet been fully realized due to several major technical and theoretical shortcomings. First, there has been a lack of systematic scheme for saturation screening in order to cover most of genes expressed in a cell and no attempt has been made for the automation of DD to ensure the reproducibility and high-throughput of the method. Secondly. it has been difficult to apply DD to systems where limited amount of samples from in vivo tissues are available. Third. there has been no efficient method for rapid and consistent recovery of the full length cDNA from the sequence information obtained by DD. without having to screen a library. Finally. no analogous method has been developed for differential display of the 5 termini of mRNAs. In this study. we propose to overcome these obstacles. (1) We provide theoretical basis and experimental support for the rational design of a set of SC arbitrary 13mers, which when used in combination with 3 one-base anchored primers will cover most of genes expressed in a cell. (2) We will adapt the Beckman Biomek 2000 robotic liquid dispensing workstation to automate the DD reaction preparations to ensure consistency and high throughput of such saturation DD screening. (3) A positive-selection cloning system will be combined with "Reverse Northern" strategy to ensure rapid cataloging and screening of the cDNA probes generated by DD. (4) We plan to further increase the sensitivity of DD towards single cell level by optimizing primer designs, isotopes and PCR conditions. (5) Using oligo-capping technique, we will develop novel methodologies for the recover of 5' full length cDNA and differential display of 5 termini of mRNAs.

描述：（申请人的描述）在估计的10万人中， 在人类基因组编码的基因中，大约10-20%由人类基因组表达。 平均细胞 这个基因子集的表达是一个主要的决定因素 一个细胞的属性。 除了控制细胞表型外， 以及癌症的正常生理过程 因此 了解这些正常和病理过程的机制 需要有机体。 基因表达的改变也是 病因学的不同病理过程，如识别。 差异表达基因的分离和表征。 差异显示（DD）技术是近年来发展起来的 并已成为最常用的识别方法， 差异表达基因的克隆。 尽管用途广泛， 由于几个主要的技术问题，DD的功能尚未完全实现 理论上的缺陷。 首先，缺乏系统的 饱和筛选方案，以覆盖大多数表达于 一个细胞，并没有试图为自动化的DD，以确保 该方法的重现性和高通量。 其次。 已经 很难将DD应用于其中来自于 体内组织是可用的。 三分之 还没有有效的方法 从序列中快速和一致地回收全长cDNA DD获得的信息。 而不必筛选图书馆。 终于来了 还没有开发出类似的方法用于5 mRNA的末端。 本研究 我们建议克服这些障碍。 (1)我们提供 为机组的合理设计提供了理论依据和实验支持 的SC任意13聚体，当与3个单碱基组合使用时， 锚定引物将覆盖细胞中表达的大多数基因。 (2)我们将 调整Beckman Biomek 2000机器人液体分配工作站， 自动化DD反应制备，以确保一致性和高 这种饱和DD筛选的通量。 (3)正选择 克隆系统将与“反向北方”战略相结合，以确保 快速编目和筛选由DD产生的cDNA探针。 (4)我们计划进一步提高DD对单细胞的敏感性， 通过优化引物设计、同位素和PCR条件， (5)使用 寡帽技术，我们将开发新的方法， 5'端全长cDNA的差异显示和5个末端的差异显示。