75. High-Throughput Multiplexed Fluorescent-Labeled Fingerprinting of BAC Clones 

Yan Ding¹, Martin D. Johnson², Wang Q. Chen³, Gigi E Park¹, Yujin Chen¹, and Hiroaki Shizuya^1 
1Beckman Institute, Division of Biology, California Institute of Technology, Pasadena, CA 91125, U. S. A; ²PE Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404, U. S. A.; and ³Paracel Inc., 80 S. Lake Ave #650, Pasadena, CA 91101-2616, U. S. A. 
yding@cco.caltech.edu 

Human Genome Project has entered in a large scale sequencing stage. Currently, numerous maps, for example, STS or EST content maps, and YAC contig maps, have been constructed across all human chromosomes. However, building of physical maps that involve large contigs of sequencing units such as BACs still falls far behind. In order to fill this gap in a timely fashion, we have been worked on high-throughput contig assembly of BAC clones through multiplexed Fluorescent-labeled fingerprinting. Projects that rely on restriction fragment size lists to establish relationships between clones require extensive overlaps due to the limited resolution inherent in these strategies. We are currently developing a fingerprinting method using certain class IIS restriction enzymes, which cut DNA a few basepair away from their recognition sites and generate 5' overhangs consisting of 1 to 5 unknown bases. With the recessed strand serving as primer, these overhangs can be sequenced using modified fluorescent dideoxy terminator sequencing reagents¹. When a fifth dye is used for an internal lane size standard, each fragment can be characterized by both size and end sequence of it's terminal 1 to 5 bases. This enhanced detail greatly increases the power to detect minimum overlap. Using this method, it is theoretically possible to identify overlaps of 15% for a project with 10,000 clones. The increased information content of each fragment also assists assembling accurate overlaps and establishing minimal tiling path with fewer clones. We will report our latest effort on optimizing the fingerprinting technique, software development to interpret the data, and a test on 500 to 1000 BAC clones, which have been identified with markers located on a 20 Mb region from 16p13.1 to 16p11.2, to assemble contigs. 

¹ Brenner, S. and Livak, K. J. 1989, Proc. Natl. Acad. Sci. USA, 86:8902-8906.