SIMPLE STRATEGY FOR SEQUENCING CDNA CLONES

We describe a simple method for constructing subclones containing over lapping nested deletions from cDNA clones (both lambda phage clones an d plasmid clones). A PCR-amplified insert is partially digested with 4 -cutter restriction enzyme(s). Complete digestion of this DNA with two restriction enzymes, halving unique cutting sites at one or the other end of the amplified DNA, creates two sets of overlapping nested subf ragments . When recloned into each of two doubly cut pBluescript(R) pl asmid vectors, only the two sets of nested subfragments are produced. Minimal nested sets can be constructed by screening subclones using co lony PCR, and this set can then be used to determine the Entire sequen ce 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 redundan cy problem inherent in shotgun cloning, allows large clones to be sequ enced using universal primers only and is well-suited for automated DN A sequencing. Using this method, we successfully sequenced five cDNA c lones of five Drosophila subobscura genes.