Dave Gray's Session 3 Email Q&A
The following is the text of my email to Lisa Scheifele following our third "Build a Gene" session along with notes regarding our discussion of these points in class:
I have just a few questions for you for tomorrow. I'll send them along just to give you a heads up.
1. The restriction enzymes in the animations have appeared in pairs. Is that because one handles splitting one strand of the DNA while the other splits the other strand? Or does each handle a different part of the process? (Or perhaps this was just a feature of the animation.)
Many restriction enzymes act as "dimers" - a pair of identical enzymes each of which cuts one strand of the DNA.
2. How do they match up to the precise pattern of nucleotides they match to? (E.g. to they contain a bit of RNA to do this?)
No, they don't have matching RNA. But they are specific to a short series of nucleotide sequences. As part of this discussion, Lisa pointed out that DNA has a major and minor "groove", the double helix of empty space between the backbones. The restriction enzymes tend to bind to the bases in the major groove.
3. The term "digestion" is used with regard to restriction enzymes breaking up DNA. That's a new use of the word to me. Is that just because they break down the DNA into smaller bits? When restriction enzymes break down viral DNA in cells, are the DNA fragments useful to the cell in any way?
Yes, it's just because they are breaking up the DNA. When restriction enzymes break down viral DNA in the cell, the DNA is decomposed and the nucleotides can be reused.
4. The term "incubate on ice" is used with regard to the transformation process. That's an odd (to me) use of the word "incubation". (If a hospital offered to incubate my newborn "on ice", I'd be looking for a new hospital!) Is it used because after mixing in the CaCl, you just let the bacteria "sit and stew" for 30 minutes to stabilize the membrane, allow the CaCl to do its work and prepare the bacteria for the "heat shock"? (Sort of the reverse process of a Finnish sauna. They do the heat first and then jump into the icy water.)
Yes, it's just because you leave the bacteria sitting on ice for a time to prepare it for transformation.
- Dave Gray