NORMALIZED HUMAN CDNA CATALOGUE WITH TISSUE-SPECIFIC IDS

我们建议构建一个规范化的人类cDNA参考目录

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.