Simple strategy for sequencing cDNA clones

We describe a simple method for constructing subclones containing overlapping nested deletions from cDNA clones (both lambda phage clones and plasmid clones). A PCR-amplified insert is partially digested with 4-cutter restriction enzyme(s). Complete digestion of this DNA with two restriction enzymes, having unique cutting sites at one or the other end of the amplified DNA, creates two sets of overlapping nested subfragments. When recloned into each of two doubly cut pBluescript plasmid vectors, only the two sets of nested subfragments are produced. Minimal nested sets can be constructed by screening subclones using colony PCR, and this set can then be used to determine the entire sequence of the cDNA clone. This method requires only a single cloning step and can be generated from an insert that is amplified directly from a lambda phage clone. This procedure eliminates the sequencing redundancy problem inherent in shotgun cloning, allows large clones to be sequenced using universal primers only and is well-suited for automated DNA-sequencing. Using this method, we successfully sequenced five cDNA clones of five Drosophila subobscura genes.