They say a magician is never supposed to tell the secret behind his tricks. It’s a good thing then that I am a molecular biologist and not an illusionist. Because once I learn a good trick, I feel the need to spread it like dandelion seeds floating on a breeze.
That’s what inspired me to tell you about The Magic of Taq and TA Cloning a little while ago. And I have been inspired once again, because the magic doesn’t end there.
Here’s the secret: ligases aren’t the only enzymes that you can use to stick two pieces of DNA together.
The rabbit in the hat: Topoisomerases
DNA topoisomerases wind and unwind coiled DNA. They do so by inducing breaks in the DNA backbone and passing strands through one another before sealing the breaks. They are important enzymes in DNA replication, transcription and recombination.
But because they can join two pieces of DNA, they can also be used to ligate an insert into a vector.
How the trick works
Topoisomerase I, isolated from Vaccinia virus, specifically recognizes the DNA sequence 5’-(C/T)CCTT-3’, cuts at this sequence, unwinds the DNA and religates it again at the 3’ phosphate group of the thymidine base.
If you also happen to provide a PCR product with a complementary A residue (like the one added by Taq polymerase), then you can conveniently ligate your PCR product to this sequence.
You can perform the magic trick by using one of the commercially available kits that provide vectors or cloning arms that are covalently linked to topoisomerase.
Advantages of topoisomerase cloning
There are several advantages to using toposisomerases for cloning:
- No need for restriction digestions
- You don’t have to gel extract your PCR product (unless you have more than one band)
- Ligation by topoisomerases is extremely efficient – ligations can be performed in as little as 5 minutes. This is faster than you can thaw your competent cells!
- Vector self-ligation occurs less frequently than with traditional ligation, increasing the chances of obtaining the correct clone.
- Although originally designed for TA cloning, newer kits also work with blunt-end cloning
I told you it was magic!