'Restriction-PCR' - a Superior Replacement for Restriction Endonucleases in DNA Cloning Applications

Polymerise chain reaction (PCR) is well established as an indispensable tool of molecular biology; and yet a limitation for cloning applications continues to be that products often require subsequent restriction digests, blunt-end ligation, or the use of special linear vectors. Here a rapid, PCR-based system is described for the simple, restriction enzyme-free generation of synthetic, `restriction-like` DNA fragments with staggered ends. Any 3`- or 5`-protruding terminus, but also non-palindromic overhangs with an unrestricted single strand length are specifically created. With longer overhangs, $quot;Restriction-PCR$quot; does not even require a ligation step prior to transformation. Thereby the technique presents a powerful tool e.g. for a successive, authentic reconstitution of sub-fragments of long genes with no need to manipulate the sequence or to introduce restriction sites. Since restriction enzyme-free and thereby devoid the limitations of partial DNA digests, $quot;Restriction-PCR$quot; allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for those endonucleases involved in the cloning. Small site-specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties $quot;Restriction-PCR$quot; has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.