Long PCR facilitates concise cloning and sequencing with a minimal tiling set of templates

We demonstrate a rapid cloning and sequencing strategy for kilobase-size DNA segments using DNase I and long PCR. In a single-tube protocol, deletions were formed in a plasmid insert by two enzymatic cuts, one at a fixed site and one at random. The doubly cut molecules were recircularized to generate a library of plasmids carrying deletions of various sizes and transformed into E. coli. The plasmid inserts were directly amplified from transformant colonies by long PCR and sized on a high-resolution agarose gel. A minimal tiling set, selected from the amplified material, was used directly as templates for long-read sequencing. The system is useful for inserts up to about 3.5 kb for de novo sequencing (both strands) or 6 kb for confirmatory sequencing (one strand).