66 -- DNA ANALYZER SEQUENCING SYSTEM, BI-DIRECTIONAL The USDA Agricultural Research Service intends to procure a DNA Analyzer Sequencing System, bi-directional, with the following capabilities: 1. Infrared laser diode, which produces minimal heat, minimizes space requirements and has a long life. System shall utilize infrared fluorescence for labeling the DNA fragments (emissions higher than 700 nm), and shall be a double dye system. The emissions of the two dyes shall be a minimum of 100 nm separated to eliminate spectral overlaps. System shall have ability to use both labeled primers and a labeled nucleotide in cycle sequencing, and shall have ability to use labeled primers, tailed primers, or a labeled nucleotide in microsatellite applications. Simultaneous bi-directional sequencing (SBS) shall be possible with this system, allowing for the sequencing of both strands of a double stranded template in a single reaction. System shall have protocols utilizing a variety of enzyme kits, including Thermosequenase Polymerase, Sequitherm Polymerase, Sequenase 17 Polymerase, TAQ Polymerase and other unspecified enzymes. 2. Vertical slab-gel electrophoresis, which allows for manual proofing/verification of sequence information. System shall have ability to make use of a minimum of three different lengths of plates. These plates shall be made of borofloat glass. Precast gels shall be available. System shall be able to provide routine read lengths of 800-1000 bp on both ssDNA and dsDNA templates. An accuracy of >98.5% accuracy shall be achieved routinely on these read lengths, with a well to read separation distance of a minimum of 31 cm on one of the sets of plates, as electrophoresis theory indicates that separate base resolution extending out this far is directly related to the electrophoresis distance (Nishikawa, T. and Kambara, H., 1992). In addition, system shall have a user-controllable speed at which to collect data, allowing for sequencing speeds of up to 200 bp/sample/hour. The gels shall have the capability for loading up to 32 samples per gel, and the capability for routinely sequencing 800-1000 bp per sample with >98.5% accuracy. When doing microsatellite work, the system shall have the capacity of 96 total lanes per gel and gels shall be reusable up to three times before replacement of the gel. Each gel shall have capability to be reloaded after an hour of electrophoresis time. An "autorad" appearing TIFF image shall be the image format so that viewing the bands is comparable to those done on autoradiography films. An infrared laser diode shall be the means of excitation of the fluorescent labels, with the lifetime of this diode in excess of 40,000 hours of operation time. The detection of the fluorescent dyes shall be approximately 710 nm to 820 nm to provide a high signal to noise ratio. The laser and detector shall be self-focusing and will accommodate the use of gels as thin as .20 mm. The laser and detector shall both rest upon a scanning platform, allowing for data to be accurately captured which migrates from a variety of comb sizes and gel lengths. System shall be operated and controlled from either a MacIntosh or PC and the sequencer shall be controlled via a TCP/IP connection enabling access to data from anywhere in the world. Each sequencer shall have it's own controller which can be communicated to via TCP/IP connections and each controller shall have it's own hard drive. System shall include sequencing software with capability of communicating with any DNA sequencer via TCP/IP connection and capability of automatically transferring sample data into an alighment/assembly software and automatically perform initial assembly. Data shall be available for viewing in a minimum of three modes: traditional band images, colored SCF curves and ASCH text. Each sequenced sample shall have capability of being viewed as a raw image for the utilization of raw data analysis and verification. The Genotyping software shall utilize a gel image for the analysis. Quantitative peak profiles shall be available for analysis. Binning and desimaling shall be possible inthe analysis software and a relational database shall be included in the same software package for transfer of data from the analyzed gel to the archiving in the database. The sequencer shall have a small footprint (e.g., 55 cm x 41 cm) and shall not require special electrical hookups, operating on 120v outlets. Equipment shall not generate excessive heat or have special cooling requirements. The Li-Cor Model 4200S-2 NEN Global IR2 DNA Analyzer System (2 dye) meets these needs and is covered under several patents. Posted 03/14/00 (W-SN434260).

Loren Data Corp. http://www.ld.com (SYN# 0453 20000316\SP-0004.MSC)