STREAMLINING DIFFERENTIAL DISPLAY TECHNOLOGY

简化差异显示技术

• 批准号: 6376631
• 负责人: PENG LIANG
• 金额: $ 25.74万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376631
• 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）估计有 100,000 人 人类基因组编码的基因，大约有10-20%是由 平均细胞。 该基因子集的表达是主要决定因素 细胞的特性。 除了控制细胞表型外 以及癌症的正常生理过程。 所以 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 病因学等多种病理过程的识别。 差异表达基因的分离和表征。 最近为此目的开发了差分显示（DD）技术 并已成为最常用的识别方法 差异表达基因的克隆。 尽管其用途广泛， 由于几项重大技术问题，DD 的威力尚未完全发挥出来 和理论缺陷。 首先，缺乏系统性 饱和筛选方案，以覆盖大多数表达的基因 一个单元，并且没有尝试过 DD 的自动化来确保 该方法的重现性和高通量。 第二。 它一直 很难将 DD 应用于样本数量有限的系统 体内组织可用。 第三。 一直没有有效的方法 从序列中快速、一致地恢复全长 cDNA DD获得的信息。 无需筛选图书馆。 最后。 目前还没有开发出类似的方法来区分 5 种情况的显示 mRNA 的末端。 在这项研究中。 我们建议克服这些障碍。 (1) 我们提供 一套合理设计的理论基础和实验支持 SC 任意 13 聚体，与 3 个一碱基组合使用时 锚定引物将覆盖细胞中表达的大部分基因。 (2) 我们将 使 Beckman Biomek 2000 机器人液体分配工作站适应 自动化 DD 反应制备以确保一致性和高 这种饱和DD筛选的吞吐量。 (3)正向选择 克隆系统将与“逆向北方”战略相结合，确保 对 DD 生成的 cDNA 探针进行快速编目和筛选。 (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 条件来提高水平。 (5) 使用 寡核苷酸加帽技术，我们将开发新的恢复方法 5' 全长 cDNA 和 mRNA 5 末端的差异显示。