LONG PCR FACILITATES CONCISE CLONING AND SEQUENCING WITH A MINIMAL TILING SET OF TEMPLATES

We demonstrate a rapid cloning and sequencing strategy for kilobase-si ze 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 recir cularized to generate a library of plasmids carrying deletions of vari ous sizes and transformed into E. coli. The plasmid inserts were direc tly amplified from transformant colonies by lon PCR and sized on a hig h-resolution agarose gel. A minimal tiling set, selected from the ampl ified material, was used directly as templates for long-read sequencin g. The system is useful for inserts up to about 3.5 kb for de novo seq uencing (both strands) or 6 kb for confirmatory sequencing (one strand ).