Simple Strategy for Sequencing cDNA Clones. (September 1996)

• Record Type: Journal Article
• Authors: Zeng, Ling-Wen; Kreitman, Martin
• Is Part Of: Biotechniques. Volume 21:Number 3(1996)
• Page Start: 446
• Page End: 452
• Publication Date: 1996-09
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/96213st01
• Languages: English
• ISSNs: 0736-6205

Abstract

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.