In order to unzip DNA, I need to create three pieces of DNA that I will then attach to each other through a ligation reaction. The first piece that I will discuss is the anchor DNA.
The anchor DNA is a very versatile piece of double stranded DNA (dsDNA). From this singular piece, we can choose to unzip DNA or stretch it because of a special sequence contained in the DNA near one end. I’ll get into this a little bit later. But first a couple of questions:
- Why is it called anchor DNA? The reason is because we use this piece of DNA to attach our entire structure to a glass surface. This is the point that anchors our DNA while we pull on it for either stretching or unzipping experiments. One of the bases is designed with a digoxigenin molecule attached to it and that base is placed right at the start of the sequence. In our tethering experiments, we coat our glass with an antibody for digoxigenin (dig for short), cleverly named anti-dig, and chemistry causes the anti-dig to bind with dig. You can understand a lot about antigen-antibody interactions here.
- How can we decide between stretching and unzipping? Because of how we designed the anchor DNA, we can stretch the anchor segment by default. That means once I produce anchor DNA I can tether it and begin stretching experiments. If we want to unzip DNA, then I take the anchor DNA and cut the end off (the side opposite the dig molecule) in a digestion reaction (more on this another time). That reaction gives me a small overhang (when one side of the DNA is longer than the other). From there I can perform a series of reactions that create the DNA sequence necessary to perform unzipping experiments. Notice that the anchor end is left unchanged, and that is what enables us to perform both stretching and unzipping experiments from this one piece of DNA.
Now the third question is, How do you make the anchor sequence? For this we need to know several sequences, possibly perform some cloning, and perform a reaction known as polymerase chain reaction, or PCR.
I’m not going to go into the details of what PCR is and how it works (google searching will reveal a lot more useful information than what I’d be willing to put here), but what I will say is that PCR allows me to make millions/billions of copies of a sequence of DNA starting with just a few strands of the original sequence and some short pieces of DNA called primers.
Our original sequence comes from plasmids. For the anchor sequence I have two possible starting points: pRL574 is a plasmid that dates back to Koch’s graduate days, and about a year and a half ago I created a brand new plasmid called pALS. Both plasmids are viable options, but serve slightly difference purposes:
- pRL574 – for this plasmid we have several different sets of primers that allow us to make anchors of different lengths ~1.1kb and ~4.4kb. The 4.4kb sequence we use primarily for stretching experiments, while the 1.1kb sequence is used in unzipping experiments.
- pALS – this plasmid only produces one length which is about 4kb. But this plasmid allows us to both unzip and stretch as I described above. It also has a couple of very unique features. First, if I cut it in the right spot, I can ligate the plasmid to itself through a special adapter sequence (to be described later). Second, it contains a sequence that is recognize by nucleosomes, that we could use for more complicated experiments down the road.
So as you can tell, I have some options available to me. Normally I would just pick one plasmid to work with, but I want to work with both and figure out which may be the more viable option down the road. In my next post, I’ll link to and list the sequences needed to make the anchor construct, with some explanations as to what everything is.