RECOMBINANT ENRICHMENT BY EXPLOITATION OF RESTRICTION SITES WITH INTERRUPTED PALINDROMES - DESIGN, SYNTHESIS AND INCORPORATION OF ZERO-BACKGROUND LINKERS IN CLONING AND EXPRESSION VECTORS

A universal approach for improving the efficiency of cloning through a selective enrichment of recombinants has been developed. This was ach ieved by using novel polylinkers or multiple cloning sites (MCS) terme d zero-background linkers (ZBL). These MCS have short-cutter site(s) i ncorporated in the wobble portion of the recognition sequence of the l ong-cutter(s) in such a fashion that cloning at the short-cutter site disrupts the continuity of the long-cutter-specific sequence. Conseque ntly, digestion of the ligation mixture with the long-cutter, prior to transformation, essentially eliminates the insert negative clones fro m transformants. The usefulness of such background removal has been il lustrated through the design and synthesis of a model ZBL molecule and then its incorporation into various popular cloning and expression ve ctors. We demonstrate that replacement of the residing MCS of a vector with ZBL leads to enhanced cloning efficiency, as evidenced by the ma rked increase in the ratio of recombinants to non-recombinants. Such v ector improvement is nearly universal as zero backgrounding is an add- on feature and can be adapted to practically any vector system, withou t having to alter the essential features of the parent vectors.