John J. Dunn, Matthew Randesi and F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, New York 11973
We have developed a vector, referred to as a fesmid, for making libraries of approximately 35-kbp DNAs for mapping and sequencing. The high efficiency lambda packaging system is used to generate libraries of clones. These clones are propagated at very low copy number under control of the replication and partitioning functions of the F factor, which helps to stabilize potentially toxic clones. A P1 lytic replicon under control of the lac repressor allows amplification simply by adding IPTG. The cloned DNA fragment is flanked by packaging signals for bacteriophage T7, and infection with an appropriate T7 mutant packages the cloned sequence into T7 phage particles, leaving most of the vector sequence behind. The size of the vector portion is such that genomic fragments packageable in lambda (normal capacity 48.5 kbp) should also be packaged in T7 (normal capacity 40 kbp).
We have made fesmid libraries of several bacterial DNAs, including Borrelia burgdorferi (the cause of Lyme disease), Bartonella henselae (the cause of cat scratch fever), E. coli, B. subtilis, H. influenzae, and S. pneumoniae, some of which have been reported to be difficult to clone in cosmid vectors. Human DNA is also readily cloned in these vectors. Brief amplification followed by infection with a gene 3 and 17.5 double mutant of T7, which is defective in replicating its own DNA, produces lysates in which essentially all of the phage particles contain the cloned DNA fragment. Simple techniques yield high-quality DNA from these phage particles. Primers for direct sequencing from the ends of fesmid clones have been made.
Primer walking from the ends of fesmid clones could be an efficient way to sequence bacterial genomes, YACs, or other large DNAs without the need for prior mapping of clones. The ends of fesmids from a random library provide multiple sites to initiate primer walking. Merging of the elongating sequences from different clones will simultaneously generate the sequence of the original DNA and determine the order of the clones. The packaged fesmid DNAs are a convenient size for multiple restriction analyses to confirm the accuracy of the nucleotide sequence.
Supported by the Office of Health and Environmental Research of the U. S. Department of Energy.