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A -- CDNA LIBRARY SOL RFP-26-82HW-98 DUE 091698 POC Dawn Reichenberg, Procurement Clerk, Fax 970-229-5531. The USDA, Agricultural Research Service intends to negotiate a contract with Life Technology, Inc. 9800 Medical Center Drive, Rockville, MD for the construction of six normalized cDNA libraries from porcine and bovine tissues. Primary cDNA libraries containing at least 3 million primary clones with an average insert size of no less than 1 kilobase will be produced from mixtures of tissues germane to meat animal research. Up to 10 million primary clones will be amplified 10,000-100,000 fold by a semi-solid agarose procedure which avoids generation of clone bias. All libraries will be normalized by a proprietary technique to a Cot of 500 or greater, resulting in a 100-fold reduction in the abundance of highly expressed sequences and a library of at least 5 million clones. Normalization and quality of each library will be assessed by colony hybridization. A typiical mammalian cell produces on the order of 10,000 to 30,000 different RNAs at any particular stage of development. The repertoire of RNAs that are produced in any one cell depends upon the cell type (e.g. liver cell, lung cell, muscle cell) and on physiologic state (e.g. liver cells change gene expression in response to starvation). The class of RNA that is eventually used as a guide for the production of proteins is called messenger RNA (mRNA). Since there are some proteins that the cell requires in large quantity, the mRNAs that code for them are produced in high abundance. Some other very critical proteins are required only in much smaller amounts, which is reflected in the amount of mRNA message that is produced. Therefore, at any given time in the cell, there are some RNAs that are present in thousands of copies, and some that are present only as a very few copies. The purpose of cDNA libraries is to provide clones of the various (mRNAs) that are present in the cell, which are in a form that are amenable to sequencing or other manipulation. The way this is done is to use an enzyme called reverse transcriptase (RT) that makes a copy of the RNA that is in the form of DNA, which can be cloned into DNA vectors. Many companies will make such libraries, and some sell kits that allow you to make your own cDNA libraries. The main problem with standard cDNA libraries is that they make a relatively faithful reproduction of the RNA repertoire of the tissue from which the RNA was isolated. That is, the abundant RNAs represent a high proportion of the clones in the library, the lower abundance mRNAs make up a much smaller proportion. The goal of our project is to pick and sequence random clones from our libraries in order to discover novel bovine and porcine genes. This process will be very inefficient if we sequence a traditional cDNA library, since a high proportion of clones sequenced will be identical. A much greater efficiency can be reached if the library has been NORMALIZED, a process in which the relative abundance of all mRNAs is equalized through very high-tech methodology. This process causes the relative abundance of clones within the cDNA library tobe altered such that the percentage of high-abundance mRNA clones is reduced and the abundance of rare mRNA clones in increased. Normalization is the key to the success of the project to sequence a significant proportion of the genes expressed in specific tissues in livestock, a critical component of moving the livestock genome maps into the modern era of functional genomics. Posted 08/25/98 (I-SN241731). (0237)

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