Double-origin vectors for isolating bidirectional deletions useful in DNA sequence analysis

Cosmids containing two compatible origins of replication (oris) have been constructed for isolating bidirectional deletions in cloned DNA. The inserted fragment and the cos site are cut, respectively, with a restriction enzyme and lambda terminase to produce two linear fragments that are circularized to produce two autonomously replicating plasmids. Each plasmid contains a different portion of the insert fused to the cosL or cosR sequence. From a series of such deletion-bearing plasmids, the nucleotide sequence of the insert can be determined in both directions using cosL and cosR primers.