What is methylation sensitivity?

Methylation-Sensitive Restriction Enzymes (MSREs) play a role in the analysis of methylated DNA, as they are used to analyze the methylation status of cytosine residues in CpG sequences. These restriction enzymes, as their name implies, are not able to cleave methylated-cytosine residues, leaving methylated DNA intact.

What is Dam DCM methylation?

Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by cloning your DNA into a dam–, dcm– strain of E. coli, such as dam–/dcm– Competent E. In this case, part of the Dam or Dcm sequence is generated by the restriction enzyme sequence, followed by the flanking sequence.

How can Dam methylation be prevented?

To avoid dam methylation use a dam-deficient strain such as dam-/dcm- Competent E. coli (#C2925). For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE.

How does methylation affect DNA cleavage?

Abstract. Sequence specific DNA methylation sometimes results in the protection of some or all of a restriction endonucleases’ cleavage sites. This is usually, but not always, the result of methylation of one or both strands of DNA at the site characteristic of the corresponding “cognate” modification methylase.

Does E coli methylate DNA?

coli is completely resistant to cleavage by MboI (NEB #R0147), which cleaves at GATC sites. The table below summarizes the possible overlaps and what is known about cleavability of DNA modified by Dam or Dcm. Almost all cloning strains are Dam+Dcm+, and many are M+ EcoKI.

Is HpaII methylation sensitive?

Such enzyme pairs are used effectively to obtain overall differences in methylation between two DNA samples. The MspI-HpaII enzyme pair is most frequently used to study the methylation status of CpG islands. This PCR-based method is highly sensitive and can be carried out with very low concentration of DNA.

Why does E. coli methylate DNA?

This occurs because DNA is protected from cleavage when a particular base in the recognition site of a restriction endonuclease is methylated. The relevant base may be methylated by one of the E. coli methylases if the methylase recognition site overlaps the endonuclease recognition site.

How does methylation affect restriction digestion?

Methylation of DNA is the most studied epigenetic modification. Many restriction enzymes are sensitive to the DNA methylation states. Cleavage may be blocked, or impaired, when a particular base in the enzyme’s recognition site is modified.

What is a function of GATC methylation in E. coli?

Role in regulation of protein expression In E. coli downstream GATC sequences are methylated, promoting transcription. For example, pyelonephritis-associated pili (PAP) phase variation in uropathogenic E. coli is controlled by Dam through the methylation of the two GATC sites proximal and distal to the PAP promoter.

Is EcoRI methylation sensitive?

Although the EcoRI enzyme recognizing GAATTC is used as an enzyme that is not sensitive to methylation in MSAP, EcoRI did not digest GAATTmC sequences when cytosines at both strands are methylated [34,35].

Why are Dam methylation sites associated with restriction enzymes?

Why? Well, even though Dam methylation sites are not specifically associated with any restriction modification systems, their sequences may overlap with restriction sites, inhibiting enzymes such as ClaI or XbaI, or, conversely, activating enzymes such as DpnI.

Why are dam and DCM methylases important in cloning?

Almost all cloning strains are Dam + Dcm +, and many are M + Eco KI. For more detailed information about K methylation, consult REBASE, the restriction enzyme database. The second reason these methylases are of interest is that the modification state of plasmid DNA can affect the frequency of transformation in special situations.

How is methylation sensitivity determined for neb restriction enzymes?

The table below summarizes methylation sensitivity for NEB restriction enzymes, indicating whether or not cleavage is blocked or impaired by Dam, Dcm or CpG methylation if or when it overlaps each recognition site. This table should be viewed as a guide to the behavior of the enzymes listed rather than an absolute indicator.

What happens when DNA is hemimethylated at dam sites?

In the first instance, it has been shown that replication initiation is suppressed when plasmid DNA is hemimethylated at Dam sites (4). Dam-modified plasmids therefore replicate once in Dam – cells, and are unable to replicate again.