Cloning without restriction: with recombinase-mediated cloning systems, worry about inconvenient or absent restriction sites becomes a thing of the past.(Technology)

Cloning DNA fragments using restriction enzymes is like flying from Seattle to New York via Phoenix. You arrive at your destination, but it takes longer than necessary to get there. Restriction sites, like airline hubs, aren't always where you need them, and sometimes they aren't there at all.

Recombinase-based methods allow scientists to move pieces of DNA from plasmid to plasmid without restriction enzymes, simplifying and expediting the cloning process. It also prevents the aggravation that comes from realizing your insert contains inconveniently placed restriction sites, or just as bad, none at all.

Such systems are useful for shuttling inserts from vector to vector--for instance, to tag a protein at both the N-and C-termini, to make both fluorescent and nonfluorescent versions of a protein, or to build constructs for expression in a variety of systems. Several methods are detailed in the literature, but only three have been commercialized: Cre/lox, Flp/frt, and lambda recombinase.

CLONING GATEWAY The Gateway technology, from Carlsbad, Calif.-based Invitrogen, uses a lambda recombinase called Clonase to catalyze in vitro recombination events between sequences flanked by 25-bpatt sites. Oftentimes, those sequences are present on two separate plasmids, as when moving an insert from a starting ("entry") construct to a "destination" vector. But Invitrogen also has systems for cloning PCR fragments directly into Gateway plasmids using Clonase (that is, without restriction enzymes and ligase). Using the CloneMiner cDNA library construction kit ($1,221 for five construction reactions), users can …