Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
© Fromme and Klingenspor; licensee BioMed Central Ltd. 2007
Received: 25 September 2007
Accepted: 26 November 2007
Published: 26 November 2007
The subcloning of a DNA fragment from an entry vector into a destination vector is a routinely performed task in molecular biology labs.
We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector.
The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.
One of the most routinely performed tasks in molecular biology labs around the world undoubtedly is the subcloning of a given DNA fragment from one plasmid vector into a different one. The reasons to do so are as numerous and diverse as are the applied methods. We here describe a further such technique involving the orchestrated action of a typeII and a typeIIs endonuclease ("outside cutter") with ligase. This procedure achieves the speed of recombinase based methods without the need for unusual recognition elements in the target vector.
Results & Discussion
In three independent tests we found between 40 and 73 colonies on the kanamycin plate (indicating pEGFP-N1) and no colonies at all (in two tests) or 10 colonies (in one test) on the ampicillin plate (harbouring pGEM-Teasy). Of 10 colonies per kanamycin plate tested for the presence of the subcloned insert in pEGFP-N1 by restriction analysis all proved to be the desired product. Noteworthy the two different resistance markers on entry and destination vector are not necessary for the procedure to function but were used for the sole purpose of easy discrimination between products in our tests. Following this proof of principle the procedure can certainly be further refined.
The entry vector once constructed can only serve to subclone into one selected restriction site. Further limitations of this technique are the inapplicability for directional cloning and the dependence on absence of the utilized endonuclease recognition sites on both vector backbone and insert. We feel, however, that the speed of this single-step benchtop procedure makes up for this when routinely cloning PCR products into a variety of destination vectors all containing the same restriction site. Our group for instance is interested in cloning certain promoter elements upstream of several different reporter genes and conversely, cloning a given open reading frame behind a series of different promoters. An even broader application can be envisaged in the field of engineering improved enzymes by gene shuffling approaches or, more generally, as one instrument in the molecular toolbox enabling the construction processes that essentially constitute synthetic biology.
* Genbank AY523564 from BglII site at base position 2325 to BglII site at 3491.
** Y+/tango-buffer (Fermentas; 33 mM Tris acetate, 10 mM Mg-acetate, 66 mM K-acetate, 0.1 g/L BSA) supplemented with 5% (w/v) PEG, 10 mM DTT and 1 mM rATP in a final volume of 10 μL. Enzyme amounts: 7.5 units Esp3I, 10 units NheI, 3 weiss units T4 ligase.
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