[19] Transposon-Based and Polymerase Chain Reaction-Based Sequencing of DNAs Cloned in ƛ Phage

Transposon- and polymerase chain reaction (PCR)-based methods for sequencing DNAs cloned in λ phage vectors hold great promise for many DNA sequencing projects. Three sets of steps are involved, each of which is automatable: (1) transposon insertion, (2) direct and crossover PCR from transposon containing phage to map insertions and generate sequencing templates, and (3) linear amplification sequencing of appropriate PCR fragments. This strategy yields accurate high-resolution sequence ladders and is cost- and labor-efficient. More development is needed, however, to realize the full potential of this transposon- and PCR-based approach. The two most serious current limitations are an undersupply of Tn5supF insertions in certain target regions and an inability to reproducibly PCR amplify over long enough distances to map all insertions of interest in λ clones in one attempt. Each of these limitations will probably be overcome in the near future.