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. 2012 Aug 13;40(22):e170. doi: 10.1093/nar/gks751

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Placing all targeted alleles within one OF does not cause a bimodal distribution for recombination frequency. EcNR2 and EcNR2.dnaG.Q576A were tested for their performance in CoS-MAGE using a set of 10 non-overlapping oligos that introduce 10 premature stop codons in the first 1890 bp of lacZ. The targeted region of the genome is likely to be small enough to be frequently encompassed within a single OF in EcNR2.dnaG.Q576A. After 1 cycle of CoS-MAGE, LacZ⁻ recombinant clones were Sanger sequenced to assay all 10 alleles. Recombinations were performed in triplicate to estimate the frequency of white colonies (lacZ⁻), but sequencing was only performed on a single replicate. (A) EcNR2.dnaG.Q576A (n = 715, 5.33:1) exhibited a significant increase in the lacZ⁻:lacZ⁺ ratio compared with EcNR2 (n = 485, 1.46:1). (B) EcNR2.dnaG.Q576A exhibited an AR distribution similar to those observed with Sets 1–3 (which span 70, 85 and 162 kb, respectively). (C) Compared with EcNR2, EcNR2.dnaG.Q576A exhibited a higher mean number of alleles converted (unpaired t-test, ***P < 0.0001). For EcNR2, n = 39, and for EcNR2.dnaG.Q576A, n = 55. (D) Compared with EcNR2, AR frequencies increased for 9 out of 10 individual alleles in EcNR2.dnaG.Q576A. The alleles are represented by their positions in lacZ (e.g. ‘+61’ means that this oligo introduces a nonsense mutation by generating a mismatch at the 61st nucleotide of lacZ). Taken together, all of these results demonstrate improved CoS-MAGE in EcNR2.dnaG.Q576A compared with EcNR2, but no significant enhancement was obtained from targeting all oligos to a single putative OF.