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. 2002 Sep 1;30(17):3818–3830. doi: 10.1093/nar/gkf501

Figure 2.

Figure 2

Methylation analysis of targeted and non-targeted HhaI and HpaII enzymes. (A) Oligodeoxynucleotide methylation assays were performed to confirm double-strand methylation by Zf.M.HhaI. All reactions contained 1 pmol Zf.M.HhaI protein and 3 pmol duplex DNA as indicated in each lane. Reactions were carried out as described in Materials and Methods. The designations 5M, 3M and 5/3M, in this and other figures, refer to the oligonucleotides being pre-methylated at the target cytosine on either the top, bottom or both DNA strands respectively. (B) Methylation competition assays. All lanes contained 1 pmol Zf.M.HhaI protein and 3 pmol HhaI oligodeoxynucleotide and competitor DNA at the following levels: lane C, no competitor DNA; lanes 2–4, Zf oligodeoxynucleotide added as competitor at 1-, 5- and 10-fold molar excess, respectively; lanes 6–8, ZfHhaI oligodeoxynucleotide added similarly at 1-, 5- and 10-fold molar excess. The arrows indicate the different mobilities of the oligonucleotides used. (C) Oligodeoxynucleotide methylation assays were performed to confirm double-strand methylation by Zf.M.HpaII. All lanes contained 1 pmol Zf.M.HpaII protein and 3 pmol duplex DNA as indicated in each lane. (D) Competition methylation analysis. All reactions contained 1 pmol Zf.M.HpaII, 3 pmol ZfHpaII oligonucleotide and competitor DNA at the following levels: lane c, no competitor DNA; lanes 2–4, as lane c except for the addition of 1-, 5- and 10-fold molar excess of ZfHhaI competitor oligodeoxynucleotide (i.e. effectively zinc finger only site); lanes 6–8, as lane c but with the addition of 1-, 5- and 10-fold molar excess of non-specific (Non-Sp.) oligodeoxynucleotide.