Overview of the method.
2017-08-30T17:38:21Z (GMT) by
<p>The method is illustrated with an example for generating a precise deletion of a hypothetical gene. (A) Two overlapping “half-cassettes” are generated in separate PCR reactions (which can be run in parallel in the same PCR cycler) using one locus specific long primer “Fp1” or “Rp1” in combination with the cassette specific primers “<i>cat</i>-midR2” or “<i>cat</i>-midF”, respectively. Each PCR fragment contain one copy of the IR (yellow arrow) and DR (light blue arrrow), as well as one of the recombinogenic 5’-homology extensions. The templates (<i>Acatsac1</i> and <i>Acatsac3</i>) differ in the location and orientation of the IR sequence, which contains the gene encoding the blue chromoprotein AmilCP. (B) The two “half-cassettes” are mixed in equimolar amounts and electroporated into λ Red induced cells. For formation of a functional <i>cat</i> gene recombination has to occur between the recombinogenic ends and the chromosome, as well as in the sequence overlap between the two “half-cassettes”. (C) The structure of the semi-stable DIRex intermediate. (D) The structure of the final deletion after spontaneous excision of the DIRex intermediate (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184126#pone.0184126.s003" target="_blank">S3 Fig</a> for a possible mechanism of excision).</p>