Restriction enzymes are a basic tool in the molecular biologist’s arsenal. They’re super easy to use, and virtually essential for cloning and other applications. Restriction enzymes are also a great example of a perfect “tool” from nature that scientists have co-opted for their own use. Here are a few fun facts about restriction enzymes that you may not have heard of:
1. The first restriction enzyme to be described was HindIII, and was originally known as endonuclease R. (see: Smith and Wilcox, J. Mol. Biol. 1970)
2. Did you ever wonder why they are referred to as restriction enzymes? Microorganisms produce enzymes to chew up foreign DNA, “restricting” the genetic content to only native DNA.
3. Restriction enzymes are named from the organism they are derived from. Thus, EcoRI is from E. coli, DraI is from D. radiophilus, ClaI is from C. latum, etc.
4. The vast majority of restriction enzymes used in the lab are Type II enzymes, which bind short recognition sequences and cut within that sequence. Type IIS and Type IIG enzymes recognize short sequences too, but cut outside of the recognition sequence, to one or both sides, depending on the enzyme.
5. There are three other types of restriction enzymes, which are not commonly used: Type I, Type III, and Type IV. Type I enzymes cut randomly at sequences distal to the recognition sequence. Type III enzymes require two recognition sequences to cleave completely, and cleave outside of these sequences. Type IV enzymes recognize and cleave DNA with foreign methylation patterns.
6. Restriction enzymes with symmetrical recognition sequences bind DNA as homodimers; enzymes with asymmetrical recognition sequences bind as heterodimers.
7. The optimum temperature for the activity of most restriction enzymes is 37°C. Some enzymes, however, work best at 55°C, 65°C, or even 75°C! That’s because these enzymes are derived from extremophiles, microorganisms that live at very high temperatures under extreme conditions.
8. If biology was as simple as the textbooks say, then a single restriction enzyme would cut equally often at every potential site in a single piece of DNA. However, there have been several reports of enzymes preferentially cutting at one site instead of another, often for no discernable reason. (see, for example: Thomas and Davis, J. Mol. Biol., 1975; Forsblum, et al., Nucl. Acids Res., 1976)
9. If enzymes are not handled using their optimum conditions, you can get unexpected off-target effects, called “star activity”. Star activity can include anything from cleavage at incorrect sites to single base substitutions, and can be caused by many factors, including extended incubation, too much glycerol, or not enough magnesium.
10. What to do if your two enzymes aren’t active in any of the same buffers? Try a sequential digest: start with the enzyme whose buffer has the lowest salt content, then add salt to optimize the buffer for the second enzyme. It’ll save you a round of purification, keep you from losing too much DNA in the process!
Recommended reading:
Check out the appendices in the New England BioLabs catalog for a lot of great information about restriction enzymes.
Most experiments start with a piece of DNA—either plasmid DNA or genomic DNA. And your downstream uses for it dictate how much you need, what contaminants you can tolerate, and your extraction and purification methods. In this article, we explain the key differences between plasmid and genomic DNA extraction methods.
Microarrays are one of the most in-depth ways of determining cellular gene expression levels of thousands of genes simultaneously. They are able to help determine: Gene function and cellular processes Gene regulation and interactions Gene expression levels in different cell types and how this expression is altered by the addition of various compounds or disease…
Working with RNA? What fun! Those little, nearly indestructible RNases are everywhere – on your skin and mucous membranes, in the water and (some of the) enzymes you use, on lab surfaces, even in airborne microbes! Here are 10 ways to keep the RNases at bay, and keep your precious samples safe:
Red Pill or Blue? Carrying out science often involves many difficult decisions! I see it all the time in RNA protocols – the “gracious” option of using purified water or Tris-EDTA (TE) buffer to dissolve (or elute, if you are using column purification) RNA. When I was trained in assessing RNA using UV spectrophotometry, graduate…
When it comes to profiling miRNAs there are lots of platforms available. We discuss the pros and cons of various miRNA profiling methods to help you choose the right one for your needs.