Plant & Animal Genomes XI Conference Plant & Animal Genomes XI ConferenceJanuary 11-15, 2003
Town & Country Convention Center
San Diego, CA
Poster: High-throughput Methods
(*Co-first authors) Construction of high-quality sequence-ready maps of overlapping BAC clones is central to large-scale genome sequencing and other aspects of genome research. To facilitate genome research, the physical maps of several species, including Arabidopsis, rice, human, mouse, soybean and chicken, have been developed. However, the methods used are manual-based, low in fingerprint resolution and throughput, and cost-inefficient. Therefore, we developed an automated procedure for whole-genome physical mapping from large-insert random BACs and BIBACs by using robotic workstations, advanced chemistries, capillary sequencers, and several new computer programs. Using the automated procedure, the clones of source BAC libraries are grown in 96-format plates. BAC DNA is isolated with the Autogen 960 robotic workstation, digested with two, three, or five restriction enzymes, end-labeled with fluorescent nucleotides and fractionated on the capillary sequencer ABI 3100. The fingerprinting data are collected and edited automatically using the new computer programs. The new procedure has not only further increased the resolution of the fingerprints (0.2 base), importantly, also increased the throughput and efficiency of whole-genome physical mapping by several fold. Using the automated procedure, we could fingerprint and analyze about 2,000 BACs in one scientist working day, from BAC libraries to contigs, and develop the contigs for a whole-genome, BAC-based physical map of a large genome, such as cotton (2,200 Mb/haploid genome), within several months. The automated procedure has been tested by using the contigs of the Arabidopsis physical map previously constructed using the polyacrylamide sequencing gel-based restriction BAC fingerprinting method.