We’ve discussed the anchor DNA and the adapter duplex, but we wouldn’t be able to measure unzipping forces and shotgun DNA mapping would fail if we didn’t have any DNA to unzip. As I’ve mentioned several times in this series of posts, the entire construct is assembled through a reaction known as ligation.

For the purposes of this experiment, the reaction works as follows: an enzyme known as DNA ligase looks for compatible ends of DNA and attaches them together. In our construct those ends are the overhangs that I referred to in the other posts. And the construct is designed so that the anchor can only attach to one end of the adapter and the unzipping segment can only attach to the other end of the adapter.

Now technically we can use any piece of DNA to unzip. The catch is that we need to use a plasmid to get the overhang that we need. As I said earlier, one side of the adapter can ligate to the anchor. The other end’s overhang is created by a cut from the enzyme EarI and is specific to the plasmid pBR322 and any other plasmids that have the same multiple cloning site. For instance, for shotgun clones (which will be explained much later) we use pBluescript II, and the enzyme SapI cuts the plasmid with the exact same overhang as EarI does in pBR322.

Because of the proximity of the SapI site to the multiple cloning site, we can stick any piece of DNA into the plasmid for cloning. Then we can cut the plasmid with the unzipping insert with SapI and then ligate this long piece to our unzipping construct.

In calibration experiments, we use pBR322 to test to make sure we have unzipping. And eventually we will move up to use the pBluescript clones that I made a few years ago. Although I have a feeling I’ll be doing that all over again.

The ligation reaction that I keep referring to requires three pieces of DNA. They get fused together all in one shot, that is slightly complicated. The most crucial of which is the adapter duplex, because without it the anchor and the unzipping DNA would not attach and the reaction would yield nothing. And because of how important the adapter is, this has been the source of my troubles for the past 4 years. But before I go into that, let me tell you about the duplex.

It’s called an adapter duplex because it is actually two single stranded pieces of DNA. We call them the top and bottom strand. They are short DNA sequences manufactured from biotech companies. In the past we’ve used Alpha DNA, but I’m thinking of trying someone new. How short are the strands? The bottom strand has about 35 bases and the top is only a few bases longer. Compare that to the anchor sequence which is either 1100 bp (base pairs) or 4400 bp or the unzipping sequence which can be as long or as short as we want (but typically around 3000bp for calibration sequences).

Once our single stranded sequences arrive via mail (we call these short sequence oligonucleotides, or oligos for short), we need to bind the top and bottom strands together in a process called annealing. Most molecular biological reactions involve some kind of enzyme to help the reaction, but annealing is quite a natural process. DNA naturally wants the bases to bind to complementary bases (A-T, G-C) and even in single stranded form, the DNA will self anneal, that is bind to itself. So to get our top and bottom strands to stick together we just put them together in the same tube, heat it up to near boiling temperatures, and slowly bring the temp down so that the top and bottom strands find each other and bind. Once it’s cooled, the adapter duplex is formed and will stay that way unless heated to very high temperatures (near boiling).

There are three key features of the adapter duplex: (1) a biotin molecule, (2) a gap in the DNA backbone, and (3) two non-palindromic overhangs. The overhangs are designed to bind with a very specific sequence. One side can only bind with the overhang I mentioned in the anchor DNA, the other side can only bind with the overhang contained in the unzipping DNA. Right now that particular sequence is very specific to cutting plasmid pBR322 with the enzyme SapI (and any other plasmids that share similar properties).

The biotin is necessary for unzipping. The biotin has a high affinity for streptavidin which coats the microspheres we use for optical tweezing. Typically the biotin in our bottom adapter strand is near the start, but not at the start of the sequence. In more recent iterations, we moved it to the 5′ end completely or added a poly-A overhang with several biotin there. The reason for this is because we’ve been having issues actually unzipping, which I’ll explain in another post. The hope was that by moving the biotin we would get better tethering efficiency and better unzipping. We ended up not getting unzipping results and the tethering efficiency studies were inconclusive.

The bottom strand has both the biotin and the gap (key feature 2), which actually plays a role in the unzipping. Since the tweezers will pull on this side, the gap was designed to aid in the unzipping. Basically the gap was the weakest point in the complete DNA chain and since the microsphere is so close to it the DNA would begin to unzip from this location. The gap is actually a missing phosphorus (the yellow in the image to the right), which prevents the anchor and the bottom adapter strand from connecting to each other.  In later iterations we completely removed the first base to make the gap wider, and the poly-A tail I mentioned was also used to prevent there from being any attachment.

Ultimately I never got unzipping to work. Oddly enough, I ran experiments that verified the ligation reaction worked, but could never get the completed structure to unzip. That’s what this new set of experiments is going to attempt. But before I get to that, I need to tell you about the unzipping DNA portion!

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:

1. 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.
2. 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.