Factors that Influence Restriction Enzyme Activity
It is not uncommon to have difficulties in digesting DNA with restriction enzymes. At times, the DNA does not appear to cut at all and sometimes it cuts only partially. If the sequence is known, restriction sites can be predicted with accuracy, but in the lab, an enzyme may cut more often than it should or at the wrong sites. In some cases, these unexpected results point to a problem not related to technique – for example, the sequence you have may be incorrect, or a restriction map provided by a colleague could be in error. However, there are a number of commonly-encountered situtions that influence how well restriction enzymes cut, and it is important to be aware of these for troubleshooting.
Different restriction enzymes have differing preferences for ionic strength (salt concentration) and major cation (sodium or potassium). A battery of 3 to 4 different buffers will handle a large number of available enzymes, although there are a few that require a unique buffer environment. In all cases, a major function of the buffer is to maintain pH of the reaction (usually at 8.0). Additionally, some enzymes are more fussy about having their optimal buffer than other enzymes. Clearly, use of the wrong buffer can lead to poor cleavage rates.
Most restriction enzymes cut best at 37C, but there are many exceptions. Enzymes isolated from thermophilic bacteria cut best at temperatures ranging from 50 to 65C. Some other enzymes have a very short half life at 37C and its recommended that they be incubated at 25C.
Influence of DNA Methylation
Almost all strains of E. coli bacteria used for propagating cloned DNA contain two site-specific DNA methylases:
The practical importance of this phenomenon is that a number of restriction endonucleases will not cleave methylated DNA. A few examples relative to Dam methylation should illustrate this concept:
The take-home message here is that if DNA unexpectedly does not cut or cuts only partially, check that the enzyme in question is not methylation-sensitive.
When DNA is digested with certain restriction enzymes under non-standard conditions, cleavage can occur at sites different from the normal recognition sequence – such aberrent cutting is called “star activity”. An example of an enzyme that can exhibit star activity is EcoRI; in this case, cleavage can occur within a number of sequences that differ from the canonical GAATTC by a single base substitutions.
So what constitutes non-standard conditions? Examples that may induce star activity include:
Digestion with Multiple Enzymes
Digesting DNA with two enzymes is a commonplace task, and oftentimes the two enzymes have different buffer requirements. There are at least three ways to handle this situation:
Variability In Digestion of Different DNA Substrates
The efficiency with which a restriction enzyme cuts its recognition sequence at different locations in a piece of DNA can vary 10 to 50-fold. This is apparently due to influences of sequences bordering the recognition site, which perhaps can either enhance or inhibit enzyme binding or activity.
A related situation is seen when restriction recognition sites are located at or very close to the ends of linear fragments of DNA. Most enzymes require a few bases on either side of their recognition site in order to bind and cleave. Many of the companies that sell enzymes provide a table in their catalog that presents “end requirements” for a variety of enzymes.