DOE Contractor-Grantee Workshop Sequencing Sessions

By:Aaron Hall

Sequence Patenting and Technology Transfer. Rebecca Eisenberg (University of Michigan Law School) focused on technology transfer, proprietary rights to DNA sequences, and access to sequence databases. She pointed out that the U.S. government is uniquely situated to enrich the public domain and that, by restricting access to valuable discoveries, the current federal pro-patent policy may sometimes undermine rather than support incentives to develop new products. Eisenberg also observed that companies may not need to protect a potentially lucrative product by obtaining patents for every step of development.

Large-Scale Sequencing

Human T-cell receptor (TCR).

Leroy Hood (UWS) reported analyzing more than 685 kb of contiguous sequence from the human TCR beta locus; this amount represents the longest stretch of human sequence yet obtained. The power of large-scale genomic sequencing and evolutionary comparisons, he noted, lies in the ability to reveal crucial genomic features undetectable by cDNA analysis alone; this approach will also generate reagents needed for access to all genomic regions. The complexity of mapping the human TCR beta locus to obtain sequencing substrates emphasizes the importance of generating rigorous sequencing technologies that can handle all problematic genomic regions. [See HGN 6(2), 1-2 (July 1994).]

Hood described his group's efforts to train high school teachers to lead their students in the shotgun sequencing of small DNA fragments encoding genes. He asked investigators to send appropriate DNA fragments if they would like to become involved with the project.

Caenorhabditis elegans.

Richard Wilson [Washington University (WU) School of Medicine Genome Sequencing Center, St. Louis], who is funded by NIH, was invited to speak on his laboratory's collaboration with the Sanger Centre (U.K.) to sequence the 100-Mb C. elegans genome. Over 9 Mb of sequence from the chromosome III central region are finished, and sequencing of chromosomes II and X is in progress. Project completion is expected in 1998. The group's sequencing strategy includes shotgunning M13 or phagemid subclones from cosmids, followed by a directed-walking finishing phase. Sequence analysis is revealing a gene about every 5.1 kb, and 42% of predicted genes have homologies to previously identified genes in databases.

Production sequencing of human and Drosophila.

The successful application of a directed-sequencing strategy was described by Michael Palazzolo (LBL). The LBL production group completed 2 Mb of sequence, representing a 23-fold throughput increase in just a year and a half. The group has achieved a sequencing rate of about 120 kb/month/person and is scaling up to 170 kb/month; this would translate to over 1 Mb/month for a six-person team. (see LBL Completes 2 Mb of DNA Sequence with Directed Strategy)

Palazzolo praised collaborations among LBL, the Drosophila genome center, and University of California, Berkeley (UCB), as well as the cooperative support of DOE and NIH. DOE supports organism-independent technology development for directed and human genomic sequencing, while NIH supports Drosophila sequencing. The 120-Mb euchromatic Drosophila genome is the first to be physically mapped with single-copy-vector large clones (PACs), which have proven stable and nonchimeric. The project has generated about 1600 STSs for an average of 1 per 55 kb and has assigned to contigs over 61% of the 6000 P1 clones for an estimated 85% coverage of the Drosophila genome.

Joseph Jaklevic reported recent progress in automating scaleup of the LBL directed-sequencing strategy, which uses gel-based PCR assays extensively for robust contig mapping. Jaklevic described work on the group's 12-channel oligosynthesizer and 3- and 4-channel thermal cyclers. The thermal cyclers are being combined with Biomek and ORCA robots to reduce material handling and tracking.

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