NORMALIZED HUMAN CDNA CATALOGUE WITH TISSUE-SPECIFIC IDS

我们建议建立一个参考标准化的人类cdna目录 其中绝大多数表达的基因都应该被表达出来。 该目录将包括许多不同归一化的cDNA 来自各种人体组织和发育阶段的文库。 在这个目录中，每个库组件都有一个特征 序列标识符(组织/特定于时间的ID)，由 用于启动第一链cdna合成的寡核苷酸，这将 对每个库都是唯一的。 所有的cDNA文库都将通过定向克隆到 根据既定方案的噬菌体媒介(Soares，1993)和 单独归一化。此归一化库集合将 然后汇集并重新归一化以生成目录。因为每个人 文库将具有特征序列识别符，任何 目录中的克隆人将会被知道。 一系列的消减杂交实验，涉及每个 单个归一化文库(或它们的组合)和 将执行目录以将目录细分为多个 根据其组织和/或时间特异性的亚库 组件。值得注意的是，因为每个库都将有一个 特定的序列识别符，将有可能评估组织 (或时间)-任何消减文库的单次通过的特异性 对随机抽样的克隆进行测序。

We are proposing to construct a reference normalized human cDNA catalogue in which the great majority of the expressed genes should be represented. The catalogue will comprise a number of different normalized cDNA libraries from a variety of human tissues and stages of development. In this catalogue, each library component will have a characteristic sequence identifier (tissue/temporal-specific IDs), provided by the oligonucleotide utilized to prime first strand cDNA synthesis, which will be unique to each library. All cDNA libraries will be constructed by directional cloning into a phagemid vector according to an established protocol (Soares, 1993) and normalized individually. This collection of normalized libraries will then be pooled and re-normalized to generate the catalogue. Because each library will have a characteristic sequence identifier, the origin of any clone within the catalogue will be known. A series of subtractive hybridization experiments involving each individual normalized library (or combinations of them) and the cDNA catalogue will be performed to subdivide the catalogue into a number of sublibraries according to the tissue and/or temporal-specificity of their components. It is noteworthy that because each library will have a specific sequence identifier, it will be possible to assess the tissue (or temporal)-specificity of any subtracted library by single pass sequencing of a random sampling of clones.