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 achieved by using novel polylinkers or multiple cloning sites (MCS) termed zero-background linkers (ZBL). These MCS have short-cutter site(s) incorporated in the wobble portion of the recognition sequence of the long-cutter(s) in such a fashion that cloning at the short-cutter site disrupts the continuity of the long-cutter-specific sequence. Consequently, digestion of the ligation mixture with the long-cutter, prior to transformation, essentially eliminates the insert negative clones from transformants. The usefulness of such background removal has been illustrated through the design and synthesis of a model ZBL molecule and then its incorporation into various popular cloning and expression vectors. We demonstrate that replacement of the residing MCS of a vector with ZBL leads to enhanced cloning efficiency, as evidenced by the marked increase in the ratio of recombinants to non-recombinants. Such vector improvement is nearly universal as zero backgrounding is an add-on feature and can be adapted to practically any vector system, without having to alter the essential features of the parent vectors.