TABLE 3

Efficiency of in vitro methylation of uncut λ DNA and λ DNA cut with HinfI or HindIII by ORF27 and DNA MTase Hia5

SampleMethylation level (dpm)
Negative control (no enzyme)180
Negative control (enzyme heated at 80°C for 15 min before reaction)180
λ DNA cut with HinfI methylated by Orf27200
λ DNA cut with HindIII methylated by Orf2722,000
Uncut λ DNA methylated by Orf2720,000
λ DNA cut with HinfI methylated by Hia5a149,000
λ DNA cut with HindIII methylated by Hia5171,000
Uncut λ DNA methylated by Hia5153,000
  • a The sequence specificity of the m6A MTase Hia5 is BA (where B = C, G, or T), so it possesses the ability to methylate almost all adenine residues in DNA. We used this enzyme as a control to show that in the λ DNA cut with HinfI, there are other potential sites to be methylated by the m6A MTase.