All right. Dr. Cotton, with regard to this diagram then, could you describe--if you would utilize this diagram and the drawing you have previously made to describe how this RFLP typing method actually works?
The diagram on the board has everything numbered so I will try to relate what I'm saying to the number. So let's say you have a sample, some biological sample. The diagram illustrates either a tube of blood or a blood stain, but there could be, as I said before, a hair root, it could be a semen stain. We will just say we have some biological sample. And the first thing that happens is that the DNA is extracted from this sample. What that means is the DNA is purified from the other components of the sample that you don't care about. The DNA is in the cell and you have all these proteins. Proteins are nice, but for purposes of DNA testing we don't need them, so you are purifying the DNA away from the other cellular components which include proteins and cell membranes and things like that. That is step no. 1.
Okay. Step no. 2 on the chart says the DNA is cut into fragments by a restriction enzyme. When I said a little while ago that if you had a method of cutting out these sections where the repeat exists, that you could look at how long it was, the method is to use a protein that is called a restriction enzyme and these enzymes have the characteristic that they can locate a specific short set of base pairs. Experimentally it is predetermined--there are many restriction enzymes. There are over a hundred different restriction enzymes. And let me just give you an example so it doesn't sound so--let's just say we have a double-stranded piece of DNA and you have this series of bases, C, C, G, G, and you go down the DNA and you find another set where it happens to have C, C, G, G. If you have a restriction enzyme that recognizes this site, it would make a cut wherever it happened to see a C, C, G, G. And I am just giving you one example. There are many others.
Actually, while you are on that drawing, Dr. Cotton, perhaps you can label that "restriction enzyme."
I'm going to go back to the previous diagram. The test is set up so that you are using a specific restriction enzyme that we know will cut outside the area of the repeats, so the enzyme that is used in the test is designed so it will cut at the beginning, just before the beginning of the repeat and just after the repeat or somewhere close to that. So you purify your DNA, you add your restriction enzyme and you make these cuts. Now, the enzyme cuts many other places, so basically if you think--if you go back to the spool of thread analogy and you have rolled out your spool of thread and it is very long, you now take a pair of scissors and cut it up into a whole bunch of small pieces and among those small pieces are these two that we are interested in.
The next step--actually the next series of steps are the steps that allow you to locate those fragments, so the first thing you do, now that you've got all these little fragments, is that you use a procedure that is called electrophoresis. It is used--it has many uses. You can do electrophoresis with proteins, but in this case we are doing electrophoresis with DNA. And that electrophoresis will allow you to separate out the pieces according to how big they are, so if I had--if I had a test-tube full of pieces of DNA that were all different size, at the end of the electrophoresis it is as if I had been able to reach in and put the small pieces down here and the medium-sized pieces over here and the larger pieces down at that end.
If this jury had heard previous testimony about the use of electrophoresis to type proteins such as PGM and EAP, would this be the same tool that you are talking about in the RFLP typing process?
The picture on the diagram that is numbered 3 is the illustration of separation of the DNA fragments on an agarose gel. That illustration is pretty opaque, but let me try on my own and see if I can make it a little more graphic. The gel is about the size of--a little smaller than an 8-by-10 piece of paper. Actually it is about the size of your notebook, a little bit wider and about that long. It is about a quarter of an inch thick and it is made of agarose. Agarose comes as a powder and you boil it up sort of like Jell-O, and it goes into solution and when you pour it into a tray it solidifies so it has some of the same characteristics of gelatin, although it is not gelatin.
So you are pouring this gel into a tray and if I look at that tray from the side after I've poured the gel, this is about one-fourth inch thick here. I also use a small mold to make an indentation at one end. If I take that same gel and I draw it as if it was sitting on the countertop where I could look down on it, I would have a series of these rectangular indentations across the top of the gel. Now, on the diagram they are sort of showing you where two samples have gone in that gel, so that the red lines, the red lanes, are sort of one that represents one indentation where you could put a sample. Anyway, you have your DNA sample and the sample is placed into this indentation in the gel which is called a sample well, and the DNA moves through the gel. The DNA has a charge, it carries a slight negative charge, so if there is a positive electrode at this end and a negative electrode at this end, DNA is forced to move through the gel and the gel acts like a sieve. Short segments of DNA can move through the gel rapidly. They can work their way through this sieve pushed around by the electric current and longer--so shorter pieces will move through the gel the farthest. The longer strands of DNA cannot move as rapidly through the gel and they do not go as far, so this is the way of taking these two pieces of DNA, these are now the pieces that we are interested in. Remember, they are part of the group that we are looking at. We have these two pieces in our mass of bits. We want to see where they are. This piece is shorter than this piece, (Indicating), so in the gel the shorter piece, let's say, moves this far and the other piece cannot move as far. So now these two pieces have separated. The piece that was only nine base pairs is in a different position than the piece that was 15 base pairs.
All right. Could we, with this diagram that you have just drawn--would "electrophoresis" be an appropriate title?
All right. Mr. Clarke, I need to allow the jurors to take a comfort break at this point. Ladies and gentlemen, I forgot to mention to you this morning, I don't know if the bailiffs mentioned to you, that we have a modified court session today. I have a funeral to attend this afternoon, so we are going to go through until one o'clock and then break at one o'clock. So we will take a ten-minute comfort break at this point and we will resume again at 12:00 sharp. All right. Please remember my admonitions to you.
The gel is about the size of--a little smaller than an 8-by-10 piece of paper... It is made of agarose. Agarose comes as a powder and you boil it up sort of like Jell-O, and it goes into solution and when you pour it into a tray it solidifies so it has some of the same characteristics of gelatin, although it is not gelatin.
If you go back to the spool of thread analogy and you have rolled out your spool of thread and it is very long, you now take a pair of scissors and cut it up into a whole bunch of small pieces and among those small pieces are these two that we are interested in.
Short segments of DNA can move through the gel rapidly. They can work their way through this sieve pushed around by the electric current and longer--so shorter pieces will move through the gel the farthest.