Introduction to the Workshop
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Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Toward an Automated System for High-Throughput DNA Sequencing: 3. Fesmid Vectors for Sequencing by Primer Walking

John J. Dunn and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973

Reliable and plentiful sources of template DNAs will be required to realize the potential for automated DNA sequencing by primer walking, using primers supplied from a hexamer library [1]. We hope to use a strategy that involves sequencing directly on 40-kbp templates. The ends of a random set of 40-kbp clones from a YAC, bacterial genome, or other large DNA would provide multiple sites to begin primer walking. With sufficient coverage, merging walks from different clones could produce the sequence of the original DNA and an ordered set of clones without the need for prior mapping steps. The multiple start sites in the original DNA should also help to overcome possible problems with repeated sequences.

We are developing new vectors for preparing 40-kbp DNAs as templates for DNA sequencing. These vectors are derived from the fosmid vectors developed in Simon's laboratory [2] and are referred to as fesmids. As with fosmids, they are designed to package 40-kbp fragments of genomic DNA into lambda phage particles, which allows the high-efficiency lambda packaging system to be used for generating libraries of clones. And likewise, upon injection into the host cell, the fesmids are maintained as single copies under control of the replication and partitioning functions of the F factor, which helps to stabilize potentially toxic clones.

The unique feature of the fesmids is that the cloned DNA fragment is flanked by replication and packaging signals recognized by bacteriophage T7. Upon infection by T7, the cloned fragment is amplified and packaged into phage particles, leaving most of the vector sequence behind. The size of the vector sequence is such that any genomic fragment able to be packaged in lambda (capacity 48.5 kbp) will also be packaged in T7 (capacity 40 kbp). Phage particles containing the genomic DNA are easily isolated from the lysate and high quality template prepared from them.

A disappointment with the first fesmid vectors was that only perhaps 5-20% of the T7 phage particles contained the cloned DNA, the remainder containing T7 DNA. To increase the fraction of particles that contained the cloned DNA, the vectors were modified to contain a copy of the lytic replicon of bacteriophage P1 under control of the lac repressor. Amplification of the plasmid by induction of this replicon a few hours before 17 infection can produce lysates where perhaps 90% of the phage particles contain the cloned DNA.

[1] Kieleczawa, J., Dunn, J. J., and Studier, F. W. (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science, 258, 1787-1791.
[2] Kim, U.-J., Shizuya, H., de Jong, P. J., Birren, B., and Simon, M. I. (1992) Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res. 20, 1083-1085.