Generating recleavable, filled-in 5’ overhangs:  This trick allows the generation of another restriction enzyme cut site through “filling in” the overhangs of a previous one.

First, a cut is made with EcoRI.  It leaves 5’ overhangs, which is important because that means the shorter strand has a free 3’ hydroxyl group (and DNA polymerases require a 3’ hydroxyl to work from).

DNA polymerase I is then used to fill in the overhangs.  Now, we have fully double-stranded blunt ends.

T4 DNA ligase is then used to seal up the blunt ends, and the original restriction site is destroyed.  However, this new site can be cut with at least two other enzymes: XranI and AseI.

Star Activity:  Restriction enzymes operate at a variety of conditions, varying buffer concentrations, pH, ionic strength, or temperature levels. 

Star activity occurs when certain restriction enzymes operate in conditions that have elevated levels of glycerol, organic solvents, or divalent cations (Mg⁺⁺/Cu⁺⁺/Co⁺⁺/Zn⁺⁺). This causes enzymes like EcoRI to become less specific about their cut site: For instance, the normal EcoRI cut site is:

Under conditions that induce star activity, the effective site becomes:

This could be problematic in some circumstances, but it can also be used intentionally as a tool in recombination.