Nucleic Acids Research, 1993, Vol. 21, No. 15 3385-3390
© 1993
METHODS |
Genome sequencing on both strands: the Janus strategy
Laboratory of Genetics, University of Wisconsin 445 Henry Mall, Madison, WI 53706, USA
Received May 8, 1993. Revised June 15, 1993. Accepted June 15, 1993.
The design of large scale DNA sequencing projects such as genome analysis demands a new approach to sequencing strategy, since neither a purely random nor a purely directed method is satisfactory. We have developed a strategy that combines these two methods in a way that preserves the advantages of both while avoiding their particular limitations. Computer simulations showed that a specific balance of random and directed sequencing was required for the most efficient strategy, termed the Janus strategy, which has been used in the Escherichia coli genome sequencing project. This approach depended on obtaining sequence easily from either strand of a cloned insert, and was facilitated by inversion of the insert in the engineered M13 vector Janus, by site-specific recombination. The inversion was accomplished simply by growth on the appropriate host strain, when the DNA strand incorporated into the new single stranded phage was complementary to that in the original phage, and was sequenced by the same simple protocol as the first strand.
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