Restriction Enzymes Resource

3.5 The Effect of Site-Specific Methylation on Promega Restriction Enzymes

Heat-inactivation of restriction enzymes may be performed when a subsequent reaction can be performed in the same reaction buffer, or when the reaction will be diluted for the next application. This will eliminate the need for extra ethanol precipitations or clean-up steps. Table 3.4 lists the sensitivity of Promega's restriction enzymes to heat-inactivation.

If little or no cutting is seen with a restriction enzyme, one possibility is that DNA methylation (or lack of methylation, in the case of Dpn I) is a problem. The sensitivity of Promega's restriction enzymes to DNA methylation is summarized in Table 3.5. If the enzyme used is sensitive to methylation, check the genetic characteristics of the bacterial strain or expression system from which the DNA was purified. The interfering type of methylation may be present.

Prokaryotic Methylation

• dcm Cytosine methylase - Methylates the C⁵ position of the internal cytosine residue in the sequence 5´...CCTGG...3´ and 5´...CCAGG...3´
• dam Adenine methylase - Methylates the N⁶ position of the adenine residue in the sequence 5´...GATC...3´

Eukaryotic Methylation

• CpG Methylates the C⁵ position of the cytosine residue in the dinucleotide recognition sequence 5´...CG...3´
• CpNpGp Methylates the C⁵ postion of the cytosine residue in the trinucleotide 5´...CNG...3´ (N = any base)

Key:

s	= sensitive to this methylation
i	= insensitive to this methylation
s(ol)	= overlapping - (sensitive when restriction site overlaps    methylation sequence)
n/a	= information not available

^(a) U.S. Pat. No. 5,391,487 has been issued to Promega Corporation for the restriction enzyme Sgf I.