The use of restriction enzymes to characterize DNA has been popular since the 1970s. Today, this technique remains one of the easiest and fastest ways to evaluate DNA sequences. As with most laboratory reagents, restriction endonuclease can be fickle. Whether you are simply digesting a plasmid, or performing a complex cloning or screening procedure, there are several key factors to consider. Correct usage conditions Every enzyme is different and requires specific conditions. For example: BSA is a stabilizer for many restriction enzymes; some enzymes do not work for longer than 1-2 hours. Most restriction enzymes digest efficiently between pH 7.2 and pH 8.5, and it is important to use the appropriate buffer. In dual restriction enzyme digestions, you may need to partially sacrifice the activity of one enzyme if the optimal conditions for the two enzymes are different, take this into account when analyzing the results. Alternatively, sequential digestions can be performed using the first enzyme, gel extraction to remove buffer components, and then digestion with the second enzyme. If this method is used, it may be wise to start with multiple tubes for the same reaction, as a lot of material may be lost during gel extraction. Methylation When bacteria replicate a plasmid, they usually methylate specific CpG islands. These sequences are often targets for methylation. Restriction sites may also fail to cut due to overlapping with methylation sites. At this time, the restriction endonuclease site and the flanking sequence just happened to constitute the Dam or Dcm recognition site, and then it was methylated to block the cleavage. Therefore, this situation should be fully considered when designing the restriction site for plasmid construction. There are three different types of methylases in laboratory E. coli strains: Dam methylase, Dcm methyltransferase, and EcoKI methylase. If you want to digest a restriction site that may be methylated, you can use a methylation-incompetent E. coli strain (JM11) to propagate your plasmid. These E. coli strains cannot methylate DNA so that your restriction enzymes can cleave CpG islands. Minimize Star activity Under certain conditions, some restriction enzymes may become scrambled and digest DNA randomly, rather than at specific recognition sites. This phenomenon, known as star activity, is usually caused by long incubation periods or poor buffer conditions such as pH. Therefore, it is critical to use the required enzymes with the recommended buffers. In addition, high glycerol concentrations may lead to increased star activity. Since most enzymes and their buffers are packaged in glycerol to extend shelf life, you should dilute buffers and enzymes sufficiently. This is why many companies recommend 20 - 50µl reactions in their general procedures and provide 10x buffer. Give the enzymes some room Certain enzymes are most efficient when there are several base pairs flanking the recognition site. This is especially important if you are going to double-cut, when the two enzymes are in close proximity, or when digesting the ends of the PCR product. Each manufacturer has specific recommendations for their product, but it is generally recommended to ensure that there are at least 6 base pairs on either side of the recognition site. The easiest way to add more base pairs is through PCR primer design, trying to avoid sequences that carry the risk of primer-dimer formation. Homolysis and heterolysis enzymes Sometimes you need to use a specific enzyme whose optimal conditions are not suitable for your experimental setup. In this case, homolysozyme may help you to carry out your experiment without seriously affecting your final results. Isoschizomers are two restriction enzymes that recognize the same site but have different properties, usually from different bacterial systems. For example, both SinI and AvaII recognize the sequence G/G(A or T)CC. However, AvaII is methylation-sensitive, while SinI is not. So if you want to cut this sequence and don't want to use a methylation-insensitive E. coli strain, then SinI would suffice, but AvaII would not. Both enzymes have the same recognition site, but cleavage at different base pairs are called isoschizomers. This may help avoid star activity and methylation sensitivity. For example, both KpnI and Acc65I recognize this site: GGTACC, but cleave at different positions. KpnI cleaves bp 5:GGTAC/C, while Acc65I cleaves bp 1:G/GTACC. KpnI may show star activity, while Acc65I is more robust. Therefore, if the cutting position is not important, Acc65I may be a better choice.

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