PAG-IV Plant Genome IV Conference

Town & Country Conference Center, San Diego, CA, January, 1995.

W22
Development of a Highly Automated Strategy for Sequencing the Arabidopsis Genome Using YAC and BAC Clones

Athanasios Theologis(1), Nancy Federspiel(2), Rick Norgren(2), Andre Marziali(2), Scott Hunicke-Smith(2), Deval Lashkari(2), Peter Oefner(2), Christine Alfano(2), Joe Ecker(3) and RON DAVIS(2)
1. Plant Gene Expression Center, USDA/UC Berkeley, CA, USA
2. Stanford DNA Sequencing and Technology Center, 855 California Avenue, Palo Alto, CA 94304, USA
3. Plant Science Institute, University of Pennsylvania, PA, USA

Yeast and bacterial artificial chromosomes provide powerful tools for isolating, mapping, and sequencing large regions of the Arabidopsis genome. We have initiated the sequencing of part of the genome using non-chimeric YAC clones from chromosome 1. We plan to compare the efficiency of sequencing YAC versus BAC clones. We are using a newly developed instrument that allows the random breakage of high molecular weight DNA by point sink shearing. Sequencing clones are being picked on an automated plaque/colony picker into 96- well plates, with a capacity of 50 plates/hour. Clones are grown in 96-well plates, and high quality template DNA is produced using a DNA template purification instrument capable of preparing 1000 samples/hour. Sequence analyses are performed on ABI 373 and ABI 377 automated sequencers. We are using a prototype automated 96-well oligonucleotide synthesizer that produces oligonucleotides in a 96-well microtiter plate at about 10% the cost of commercially available oligonucleotides. We are developing other instruments with improved design and software that will be available to the Arabidopsis project. One of these is a new sequencing reaction instrument which will be much faster than the ABI Catalyst while using much less reagent. We are also developing a new YAC vector that should solve most of the chimera and instability problems, providing a more reliable source of sequencing clones. We will discuss strategies for sequencing the entire Arabidopsis genome in the most effective collaborative manner, leading to the eventual sequencing of more complex genomes of agronomically important crops.

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