U.S. Department of Agriculture, ARS, LPSI, East Side Service Center, Bldg. 307, Room 129, BARC-East, Beltsville, MD 20705

66 -- CUSTOM NORMALIZATION CDNA LIBRARY SOL 0011265120-00091 DUE 070700 POC John Wilkinson, Telephone (301) 504-5799, Fax (301) 504-8696 USDA intends to award the following solicitation as a "Sole Source Procurement" due to the "PATENT" that "Life Technologies Inc." holds for the "Superscript RT Enzyme". USDA intends to purchase a "Custom Normalized cDNA Library". This procurement request is for cDNA library normalization service to construct a normalized cDNA library from chicken intestinal lymphocytes. A normalized cDNA library of low clonal redundancy is required as a resource by our laboratory to efficiently generate 10,000 unique expressed sequence tags (ESTs) from the chicken intestinal lymphocytes. This service needs to follow specific parameters to insure our lab obtains the highest quality cDNA library necessary for our EST project. First, mRNA has to be isolated from chicken intestinal lymphocytes at day 0, 1, 2, 3, and 4 post infection of E. maxima. The mRNA must be mixed in equimolar concentrations before producing >1 kb cDNA fragments with a high fidelity enzyme like Superscript II H- RNase reverse transcriptase. USDA requires these cDNAs fragments to be cloned into pCMV Sport 6.0 vector and transformed into DH10B cells. From these reactions, two libraries should be produced; each with >1 x 107 primary clones. Individual clones from these libraries need to be analyzed to determine average cDNA insert size. The primary libraries have to be produced in 4-6 weeks. A hundred clones are then returned to our laboratory for sequence analysis to determine redundant clones for quality control analysis of the normalization procedure. The primary libraries are amplified using a semi-solid agarose procedure to minimize clonal bias that would occur in a liquid amplification. The libraries should be amplified 10-100,000 fold. Once amplified, the DNA isolated from the libraries should undergo a Cot-500 normalization using subtractive hybridization technology. The DNA is again transformed into E. coli to produce a final copy of the normalized cDNA library containing a minimum of 5 x 106 unique clones with an average insert size >1 kb.Completion of the normalized library from the primary libraries has to be done in 4-6 weeks. The resultant normalized library should have reduced abundant genes by 10-100 fold. Levels of ribosomal RNA clones also need to be determined by colony hybridization. All steps used to produce the cDNA library must be assured by a quality control process. All residual materials need to be returned to our laboratory. A copy of the solicitation will be made by WRITTEN REQUEST ONLY. This solicitation incorporates clauses that are in effect through Federal Acquisition Circular 97-17. This acquisition will be solicited on a unrestricted basis. The Standard Industrial Classification Code is 8731. Delivery is required 30 days after award of this solicitation. Delivery shall be F.O.B. Destination to the U.S. Department of Agriculture in Beltsville, Maryland. Any interested/qualified sources who can provide the item stated above, must respond by July 7, 2000. All responses will be evaluated in order to determine the capabilities stated in the RFQ. The Government will award a contract resulting from this solicitation to the responsive/responsible offerer whose quote conforms to the solicitation and will be most advantageous to the Government based on price. This procurement shall be conducted under FAR Part 12, Commercial Items. Posted 06/20/00 (W-SN466460). (0172)

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