Enzyme FET

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. Buffer Composition 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. Incubation Temperature 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: Dam methylase adds a methyl group to the adenine in the sequence GATC, yielding a sequence symbolized as GmATC. Dcm methylase methylates the internal cytosine in CC(A/T)GG, generating the sequence CmC(A/T)GG. 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: MboI and Sau3AI are isoschizomers that recognize and cleave the sequence GATC, which is precisely the sequence recognized by Dam methylase. Digestion of GmATC by MboI is completely inhibited, while digestion by Sau3AI is unaffected by methylation. The recognition site for ClaI is ATCGAT, which is not a substrate for Dam methylase. However, if that sequence is followed by a C or preceeded by a G, a Dam recognition site is generated and cleavage by ClaI is inhibited. Thus, arandom sequence of DNA propagated in most strains of E. coli, half of the ClaI recognition sites will not cut. 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. Star Activity 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: High pH (>8.0) or low ionic strength (e.g. if you forget to add the buffer) Glycerol concentrations > 5% (enzymes are usually sold as concentrates in 50% glycerol) Extremely high concentration of enzyme (>100 U/ug of DNA) Presence of organic solvents in the reaction (e.g. ethanol, DMSO) 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: Digest with both enzymes in the same buffer. In many cases, even those a given buffer is not optimal for an enzyme, you can still get quite good cleavage rates. Enzyme manufacturer catalogs usually contain a reference table recommended the best single buffer for conducting specific double digests. Cut with one enzyme, then alter the buffer composition and cut with the second enzyme. This usually applies to situations where one enzyme like a low salt buffer and the other a high salt buffer, in which case you can digest with the first enzyme for a time, add a calculated amount of concentrated NaCl and cut with the second enzyme. Change buffers between digestion with two enzymes. In some cases, two enzymes will have totally incompatible buffers. In that case, perform one digestion, recover the DNA (usually by precipitation) and resuspend in the buffer appropriate for the second enzyme. 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.

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