HIGH-THROUGHPUT FINGERPRINTING OF BACTERIAL ARTIFICIAL CHROMOSOMES USING THE SNAPSHOTTM LABELING KIT AND SIZING OF RESTRICTION FRAGMENTS BY CAPILLARY ELECTROPHORESIS.

Authors: LUO, MING-CHENG - UC DAVIS, CA; THOMAS, CAROLYN - UC DAVIS, CA; YOU, FRANK - UC DAVIS, CA; HSIAO, JOSEPH - GEN RES, ROCKVILLE, MD; OUYANG, SHU - GEN RES, ROCKVILLE, MD; BUELL, ROBIN - GEN RES, ROCKVILLE, MD; MALANDRO, MARK - SAGRES, DAVIS, CA; MCGUIRE, PATRICK - UC DAVIS, CA; Anderson, Olin; DVORAK, JAN - UC DAVIS

Submitted to: Genome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2003
Publication Date: 10/1/2003
Citation: Luo, M., Thomas, C., You, F.M., Hsiao, J., Ouyang, S., Buell, R.C., Malandro, M., Mcguire, P.E., Anderson, O.D., Dvorak, J. 2003. High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot(tm) labeling kit and sizing of restriction fragments by capillary electrophoresis. Genome Research. 82:378-389.

Interpretive Summary: Although wheat is one of the most important crops worldwide, a major difficulty in the application of modern genomics technologies to wheat is that the genome of wheat is large; 40 times the size of the rice genome, and five times larger than corn. Without the application of such technologies, the full application of the most modern techniques for wheat improvement will be retarded. This paper reports a highly efficient method of determining a key feature in wheat genomics; i.e., the actual order and isolation of the genes of wheat. This procedure uses capilliary glass tubing to separate DNA fragments and computer software to assemble the structure of the wheat genome. Applications of this technology to future experiments in understanding wheat and its improvement are discussed.

Technical Abstract: An automated, high-throughput fingerprinting technique was developed that makes it possible to rapidly fingerprint large numbers of bacterial artificial chromosomes (BAC) clones, automatically edit the resulting fingerprints, and assemble contigs. BAC DNA was isolated in a 96-well plate format, simultaneously restricted with four 6-base pair (BP) recognizing restriction endonucleases that generate 3' recessed ends and one-4-bp recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends was labeled with a different flourescent dye and estriction fragments were separated on a capillary DNA sequencer. The accuracy of the technique was demonstrated by reconstruction of contigs of rice BAC clones with know positions on rice chromosome 10.