We talked about inserting passenger into plasmids last time. Briefly, the process consisted of cutting the vector and the passenger with the same restriction enzyme. This produced the same sticky ends on both molecules and allowed them to be ligated together with the help of the enzyme, DNA ligase.
This method of cloning has been used for a long time, but it has several disadvantages. For one thing, when a circular vector is cut with a specific restriction enzyme, the two ends of the now linear molecule can rejoin, without addition of a passenger. For another, the passenger can also circularize.
Many other reactions are also possible, all of them alternatives that interfere with joining one passenger with one vector. You might list those that you can think of. But even if everything goes fine and a single passenger molecule combines with a single vector, the passenger can join together in two orientations (How?). Sorting through these multiple possibilities takes time and costs money. For increased efficiency, other methods have been diligently searched for.
In our particular case, we want to clone a piece of DNA that has been amplified by polymerase chain reaction. Cloning PCR products can be a challenge. For example, PCR products often have one or more A residues on the ends of the primers put there by Taq polymerase. This results in ends that are not blunt, but not particularly sticky either, making ligation difficult.
A few years ago, Stewart Shuman, who is at Sloan-Kettering Institute in New York, devised a new way of inserting PCR products into plasmid vectors. He apparently patented the process and sold or licensed it to "inVitrogen", a biotech company that has its headquarters in California. The process makes use a single enzyme, topoisomerase I from Vaccinia virus, to both cut and ligate DNA.
The Enzyme
Topoisomerases in general are enzymes that untwist DNA. Think of DNA as two rubber hoses (each hose can be envisioned as one DNA strand) twisted around each other. An electron micrograph of this twisted DNA is shown above. During replication and transcription, the two strands must be separated from one another. That separation requires untwisting. If you untwist a closed circular double-stranded molecule, it will overwind (called technically "supercoiling") at another position. The topoisomerase enzymes prevent this overwinding.
The topoisomerase from vaccinia virus recognizes a specific sequence (CCCTT) on double stranded DNA and binds to this sequence (remember, enzymes recognize and bind; that's what they do). This topoisomerase then cuts one strand of the DNA at the last T. By cutting the DNA, the overwinding pressure can be relieved. Then, after cutting the DNA and unwinding it, the enzyme reforms the covalent bond between nucleotides. In technical terms, it ligates the ends that it had cut back together.
Invitrogen takes advantage of the cutting and ligating. It provides a plasmid in which the topoisomerase has cut the DNA and left a one base "T" overhang. Because PCR often leaves a single A residue on amplified products, the two ends base pair, and the topoisomerase knits them together. It turns out it does this with great specificity and efficiency.
