The PCR reaction

Today we will talk about a pretty important biochemical reaction used in many biochemistry labs for many different things. Basically, the PCR reaction lets you obtain lots of DNA from a very small sample. That's why you heard it over and over during OJ's trial, and why many people makes the connection between PCR and forensic science.

PCR, or the polymerase chain reaction, is a fantastic tool for those working in DNA, particularly for those fishing for minute ammounts of genes in a cell. The process was developed by Jary Mullis, who won the Nobel Prize in Chemistry for his invention in 1993. It uses the complementarity principle of DNA and DNA replication to amplify the concentration of a certain part of a DNA double-helix strand by several billions.

Say that you found a bloody glove, but the DNA you got from the sample was not enough to do genetic fingerprinting. The first step in PCR is to find two sections of the DNA that you know should exist. This can be determined by some common feature that does not change from the DNA of one person to the other, as it for example the DNA that encodes for certain enzymes (hemoglobin should be the same in everyone, for example).

Second, you have the DNA lab prepare two short strands of DNA that are complementary to one side of these two sites you found. These are called the DNA primers. They don't need to be more than 20 bases long.

Now you have your primers, and your DNA sample from which you want the stuff in the middle of the two sections described above. The first thing you have to do is to unwind the DNA double-helix. In the lab, you do not have the enzyme that does this in the organism, called helicase. As you know, the forces that keep DNA together are h-bonds between the complementary bases, so if you increase the temperature enough, you should be able to get single stranded DNA:

Now you add your primers, which will be in high concentration with respect to the original DNA. At this point, you start cooling the solution slowly, so that it does not colapses rapidly. This is called annealing of the DNA. Since there is a lot more of the primers than there is of the origina DNA strands, they will base pair to the appropriate locations they are complementary (remember, you designed them this way...):

One this that we did not mention is that we have a polymerase in the solution. Upon addition of deoxynucelotide triphosphates (dATP, dGTP, dCTP, dTTP), this polymerase will start making chains of new DNA starting from the primers, complementary to the original DNA strands we had:

Now, this things will grow as long as the polymerase does not fall off the DNA strand, and as long as we have dNTPs around, making two new strands that have the section we were looking for. In the next step, we heat the DNA solution again in order to separate the new double-helices that formed:

One proble of doing this over and over is most polymerases go to pieces with heat. Unless we want to add polymerase every time, we have to use a polymerase that can take the beating. We use the polymerase from a bug that lives in water at 80^oC (geysers and hot springs). The bug is thermus aquaticus, and the polymerase is called Taq polymerase. At this point, we add more primer (actually the primer is there already. We let the DNA anneal, and we will have again complemenrarity between the primers and the DNA strands. Note that in this case, two of the strand come from our first PCR cycle, so they will finish at the primer:

We add more dNTPs (again, they are usually already there), and we just let Taq polymerase work another cycle:

In this cycle, we have four new strands made. Two of them look like the ones made in the first cycle. The other two are just the lenght of the DNA fragment that we were interested, the one between the primer complementary sites. Now we do the same thing again, heat, then cool, then heat, then cool...

In the second cycle, we will get one double-helix that has the stuff we are interested in. In the third, two; In the fourth, four; in the fifth, eight; in the sixth, sixteen; etc., etc. The increase in the ammount of the DNA we want is geometric. After 22 cycles (2²⁰), you usually have enough concentration of the DNA fragment you want to do whatever you want, and hopefully find whose blood was in the bloddy glove.

That's all folks! No more classes, just finish the take-home, and get ready for final. Hope you enjoyed it, and if you did not, hope it wasn't that bad. Have a very nice Winter Holiday!

Prepared by Guillermo Moyna, 1999.