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.