Wednesday, April 14, 2010

My Research Explained: The Unzipping Construct

Last time I discussed the possibilities that arise when you mix DNA with restriction enzymes. Today I'm going to talk about an application very specific to my research, the creation of the unzipping construct:

After construction of the DNA structure you see above I have one continuous piece of double stranded DNA which will be pulled apart by the optical tweezers. But before we get to that stage a lot of planning, preparation, and perfect execution must be done. There are three main components in the construct. There is:
  1. The anchor segment - this piece of DNA binds the entire construct to a glass surface via antibody-antigen interactions and is created from a process known as Polymerase Chain Reaction (PCR) both of which are complicated enough to get their own posts.
  2. The adapter duplex - this piece of DNA has 3 very important features: it joins the anchor to any piece of DNA that we wish to inspect, it has a biotin (which binds to streptavidin which is attached to microspheres that we apply forces to with our tweezers), and it has a single small gap between two base pairs. It is this gap that allows us to easily unzip DNA.
  3. Inspection DNA - this is the DNA that we will ultimately get unzipping signatures from. This is the DNA that we study and can manipulate for all sorts of experiments (also to get it's own post).
We are able to combine these three individual pieces with the aid of DNA Ligase, a ligation enzyme, which can attach complimentary DNA (remember the sticky ends I talked about last time). Each segment has a unique sticky end which I will talk about in a moment.

In the construct, the anchor segment is always the same. By this I mean that no matter what piece of DNA I want to study, it will be attached to the same anchor segment (just a copy). Sometimes I will be able to use the same adapter duplex as well, but not always. Don't worry, it is easy to change the adapter because the adapter is actually made from two single stranded pieces that are complimentary. One piece is shorter than the other and binds to the middle leaving two overhangs. So if I change an end of the longer strand (not the piece that has the compliment of the anchor) I can attach a different piece of DNA that I would like to unzip. Easy! (Not really...) I am able to combine the two single strands into one piece of double stranded DNA because thermodynamics is a wonderful thing. I simply put my DNA in a little tube and heat it up. As the solution cools, the DNA will automatically create the formations that I require for my experiment. It literally only takes the amount of time to let almost boiling water cool to room temperature.

We are just about ready to perform our ligation. We have our adapter duplex and our unzippable DNA. We just need the anchor construct. The PCR reaction creates double-stranded DNA but leaves a blunt end (no sticky end or overhang). We need to create this overhang so I digest my anchor DNA with an enzyme called BstXI which leaves a very unique overhang. So unique that only two things bind to it, the adapter DNA and the rest of the anchor (remember we cut the single piece of DNA into two pieces). So I purify the piece I need and now I'm ready.

I forgot to mention that in recent experiments my unzippable DNA needs to be digested with a different enzyme, SapI. This also leaves a unique overhang so that only the other piece from the cut site binds to it or the other end of the adapter duplex (remember I mentioned that the adapter has two overhangs and one end will bind to the anchor). I can design the adapter to have any overhang I want but would normally only want to use enzymes that provide unique overhangs and nonpalindromic overhangs (CTCGAG is palindromic because the complimentary strand is identical and thus this sequence could fold up so the CTC binds to the GAG on the same strand!).

Anyways, taking all that into consideration we are now ready for the next step, the ligation. (I told you there was a lot to plan out and prepare.) In short, I mix the enzyme, some buffer solution, and my three DNA segments into one solution. I let the reaction play out at 16C and then separate the various products of the reaction using gel electrophoresis (to be explained in another post). If everything works out ok I should see something that looks like this:Which you can find originally from here.
The second column of bands (the bright lines) is what we want to see (the other lanes are extraneous information). You see three bands in that column. The band on the bottom of the image is due to unbound anchor DNA. The next band up is from unbound unzippable DNA. We don't see a band from unbound adapter DNA because that sequence is extremely small compared to the other two. Finally the third band at the top is the successful combination of the three DNA segments I put in the reaction. I simply purify that one piece of DNA (which has a lot of identical copies of the construct in it) from the rest of it and I'm ready to do stuff like this:Next time I will talk about two important staples of molecular biology: PCR and Gel Electrophoresis. I might separate them into two separate posts if it gets lengthy. The time after that I will talk about how I am able to rely on the bonds I discussed in this post (streptavidin-biotin and antibody-antigen).

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