A pBR322-derived Vector for Cloning Blunt-ended cDNA: Its Use to Detect Molecular Clones of Low-abundance mRNAs

Abstract

In place of the unique Pst I site in pBR322, we have engineered by GC tailing a unique Sma I site bracketed by Pst I sites. The resulting vector, pDE61, and an improved derivative with greater symmetry around the Sma I site, pDE613, have been used to clone blunt-ended duplex cDNA molecules in Escherichia coli in an efficient manner (5 × 10⁵ clones from 1 μg of double-stranded cDNA). When DNA is cloned into the Sma I site, the ability of both vectors to confer ampicillin resistance is lost. Evidence suggests that functional β-lactamase is made only after the GC-rich sequence containing the Sma I site is deleted: an insert in the Sma I site prevents this. Libraries in either vector, with single or multiple inserts, can be used to generate amplified amounts of cloned heterogeneous cDNA for screening other "target" libraries in a non-homologous vector (e.g., a Bacillus subtilis vector) for cDNA clones of low-abundance mRNAs. Species as infrequent as 0.003% can be readily detected by colony hybridization.