Figure 3

Exploration of ssOR by alterations in oligo design. The contribution of the oligo to ssOR was explored by altering the oligos in three different ways. A. The position of the repair site was shifted from a central position in 50 mers (23/23) to more 3' (35/11) or 5' (11/35), as well as at the 5' (0/20; 0/100) or 3' (20/0; 100/0) terminae, for both lagging and leading oligos. Results are from a single experiment performed with competent cell batches made in parallel after induction of RecT or Redβ expression. B. The ability of ssOR to repair larger deletions in the template was evaluated with three further derivatives of pGKneoΔ that lacked 15, 33 or 60 bps around the NcoI site of the kanamycin resistance gene. Oligos that retained 22 nucleotides of homology either side of the the 4, 15, 33 or 60 nucleotides required to restore kanamycin resistance were co-electroporated with pGKneoΔ. Results are from a single experiment performed with lagging oligos and competent cell batches made in parallel after induction of RecT or Redβ expression. C. The impact of a second mismatch in the oligo was evaluated using 92 mers with the NcoI site off center leaving a short side of 29 nts from either the 5' or 3' end. The longer side of 59 nts was interrupted either by a point mutation 14 or 29 nts from the NcoI site. Results shown are from a single experiment using a lagging oligo. Qualitatively similar results were obtained with the corresponding leading strand oligos (data not shown). The two, rare, events in which the point mutation was also incorporated were found with the 44/14/29 oligo illustrated.