Open AccessSimple Strategy for Sequencing cDNA Clones
Author(s) -
Lingwen Zeng,
Martin Kreitman
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96213st01
Subject(s) - biology , insert (composites) , complementary dna , restriction enzyme , genetics , cloning (programming) , restriction site , microbiology and biotechnology , clone (java method) , multiple cloning site , molecular cloning , plasmid , restriction digest , lambda phage , dna , gene , bacteriophage , recombinant dna , escherichia coli , vector (molecular biology) , mechanical engineering , computer science , engineering , programming language
We describe a simple method for constructing subclones containing overlapping nested deletions from cDNA clones (both λ phage clones and plasmid clones). A PCRamplified 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 λ 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.