STREAMLINING DIFFERENTIAL DISPLAY TECHNOLOGY

描述：(申请者的描述)约100,000人 由人类基因组编码的基因，大约10%-20%由 平均单元格。这部分基因的表达是一个主要的决定因素 一个细胞的属性。除了对细胞表型的控制 以及癌症的正常生理过程。因此 对这些正常和病理过程的机制的理解 需要有机体。基因表达的变化也是导致 病原学鉴定等多种病理过程。 差异表达基因的分离和鉴定。 差异显示(DD)技术就是为此目的而发展起来的 并已成为最常用的识别和识别方法 差异表达基因的克隆。尽管它被广泛使用，但 由于几个主要的技术原因，DD的强大功能尚未完全实现 和理论上的缺陷。首先，缺乏系统性的 饱和筛选方案，以覆盖在 一个单元格，并且没有对DD的自动化进行尝试以确保 该方法重现性好、产量高。其次。一直以来都是这样 很难将DD应用于以下情况下的系统： 活体组织是可用的。第三。目前还没有有效的方法来解决 从该序列中快速、一致地回收全长c DNA 由DD获得的信息。而不必对图书馆进行筛选。终于来了。 还没有开发出类似的方法来差异显示5个 MRNAs的终端。 在这项研究中。我们建议克服这些障碍。(1)我们提供 机组合理设计的理论基础和实验支持 当它与3个一碱基结合使用时， 锚定的引物将覆盖细胞中表达的大部分基因。(2)我们会 将Beckman Biomek 2000机器人液体点胶工作站改造为 自动化DD反应准备，以确保一致性和高效性 这种饱和DD筛选的吞吐量。(3)正面评选 克隆系统将与“反向北方”战略相结合，以确保 DD产生的cDNA探针的快速编目和筛选。 (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、同位素和聚合酶链式反应条件。(5)使用 寡头封顶技术，我们将开发新的恢复方法 5‘全长cDNAs和mRNAs 5末端差异显示。

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.