RECOMBINANT DNA 2
Lecture 16
Good morning. So, we are going to see if my voice holds up through this lecture today. It is a casualty of having been at Foxborough yesterday, and then staying up rather late watching the Red Sox game. On the whole, both seemed to have come through successfully, but my voice is a bit of a casualty of the events. So, we'll see. But I'm going to sound a lot scratchier than normal.
So, how many of you stayed up to the end of the last night? Good, excellent. I approve.
OK,last time, we spoke about the idea of cloning DNA, to create libraries of molecules.
And again, I think this is just one of the most clever inventions because it's a completely new way to think about purifying molecules. Rather than purifying molecules, by separating them based on their biochemical properties, it's purifying molecules by diluting them into single components, and then amplifying each back up from its own source. It's really quite a beautiful idea. And just to go over it, we take, say, human DNA, or we could take drosophila DNA, or we could take yeast DNA, or we could take any other DNA we feel like.
We cut it up in some fashion with a restriction enzyme. We'll use our favorite restriction enzyme here, EcoRI, which cuts at a defined site, GAATTC. We take that. We add our insert DNA. These are referred to as inserts because they're going to be inserted into a plasmid.
We take a plasmid vector. The plasmid vector here is a naturally occurring, although sometimes modified, piece of DNA that bacteria have that take an origin of replication that allows it to grow autonomously when put in a bacterial cell, a selectable marker.
The selectable marker, for example, ampicillin resistance, or some other resistance, we add these and then we seal up the pieces of the DNA using the enzyme ligase. Ligase joins and joins, producing for us molecules of this sort. We make zillions of them in parallel in one test tube. We then transform them by adding these molecules to bacterial cells that have been appropriately prepared to be transformed, that is, their membranes have been treated in such a way that they're going to be most likely to suck up pieces of DNA.
We then plate them on a plate at a density so that individual bacterial cells are well separated from each other. You try a bunch of different densities so you get one right. And, you let them grow up. And, every colony here, as we discussed, is the descendant of a single bacterial cell, carrying ideally a single plasmid.
And, that single plasmid, we know it's carrying a single plasmid because we were clever enough to put ampicillin or another selectable marker on this plate. And so, only bacteria that have picked up the plasmid are ampicillin resistant. And there you go. This is called a library. And, at the end of the day, you may have a library that contains one plate of clones or a library containing hundreds of plates of clones.
We're going to see how we last through this. Now, a few people asked me at the end of the last lecture, well, OK, but what about the details. Is it really going to work like this? How come some of these plasmid molecules don't automatically get closed back up by ligase? Why is it that there's always an insert in the plasmid?
What's the answer to that question? Sorry? There's not an answer because sometimes ligase might close up that molecule. Now, that would be unfortunate because it would mean that a bunch of the things in your library just had the vector without any insert. So, and these are details, but over the course of years, recombinant DNA specialists have worked out lots of cute tricks to make better and better libraries. I'll just give you an example of the kinds of things.
Remember that in order to ligate DNA, we had a five prime here. We have a phosphate group here, three prime hydroxyl phosphate here, double strand of DNA here. We have a phosphate here. We have a hydroxyl here, phosphate five prime, three prime.
