Enrichment PCR detection of ZFN-mediated NHEJ and GT at the ADH1 locus. (A) Detection of mutagenesis at the ADH1 locus. Protoplasts derived from the five genotypes were treated with estradiol. An enrichment PCR procedure (see Methods) was performed to detect ZFN-induced mutagenesis (uncut band). This experiment was repeated twice with similar results. (White asterisks) PCR products shorter than wild type (WT). (B) Simultaneous detection of mutagenesis and GT at the ADH1 locus. Protoplasts derived from the five genotypes were treated with both estradiol and the donor. An enrichment PCR procedure was performed to detect ZFN-induced mutagenesis (lower uncut band) and GT (upper uncut band) (red asterisk). (White asterisks) PCR products shorter than WT. Note that for one of the two WT controls (lane 1), the first round of NlaIII digestion of the WT DNA was so complete that very little PCR product was obtained for the second round of digestion. This experiment was repeated twice with similar results. (C) DNA sequence confirmation of gene targeting events. The larger PCR band indicated by a red asterisk in panel B in the ku70 background was cloned and sequenced. The upper box indicates a sequencing trace expected for an HR product. The lower box indicates the 12 bp in the WT target locus that was deleted through GT.