PCR

Finally, I can sit down to have my heart-attack. For the last 1.5 hours or so, I have been setting up PCR, or the polymerase chain reaction. This is the first step in a much larger process and the first big thing that I have done by myself.

PCR is a very complicated process. The premise is to be able to copy (many, many times) a piece of DNA. There is a particular segment of DNA that you want to study, or in our case you want to study the proteins that bind to that DNA.

A bunch of reagents get mixed together. These include primers, dNTPs, polymerase, buffer, and of course the DNA template. There are forward and reverse primers, each one travelling in one direction along the DNA strand. They are complementary to the ends of the template. dNTPs (deoxynucleoside triphosphates) are the building blocks of DNA. Polymerase is the stuff that actually does the copying/making new DNA.

After mixing these together in the right proportions, which is the only thing I was actually doing for the past hour and a half, they are put into tiny thin-walled test tubes called PCR tubes. This is so that their temperatures can be changed very quickly. There are 50 micro-liters in each tube (about 1 drop of water's worth). These get put into a thermocycler. This is a device that is able to change temperature very quickly, and sustain a certain temperature for a given amount of time. Each temperature allows for different parts of PCR to occur.

First, the mixture is heated a lot. In our case, 94 degrees C. This is to denature the DNA, ie. to make it melty. It separates the bonds of the DNA that would normally make it a double spiral. It is now two single strands.

The temperature now goes down to 55 degrees C. This is the optimal temperature for the primers to attach to the strands. Remember how there is a forward and a reverse? The primer attaches in the middle of the strand, so this is necessary. Also, it can only do reverse in short segments, and these have to get zipped together at the end. Thus, the forward primers need to circle back. The polymerase will now attach to the primer-template hybrid. This is called annealing.

The temperature is raised to 72 degrees C. This is the optimal temperature for the polymerase to make new DNA. The primer is sort of a blueprint for the polymerase to know how/which dNTP to attach to make DNA. This is the extension step.

This process is one cycle. With each cycle you have double the amount of DNA material. We repeat this 45 times. I think that means we end up with 2^45 times the amount of DNA than we originally had as the template. Regardless of the math, it's a tremendous duplication.

At the end, it gets chilled to 4 degrees C, and it can stay there indefinitely. My PCR wont be done before the workday is over, so I can fetch it tomorrow.

I did this all by myself. *pats self on back, and says, "I am terrific."* AB wasn't even looking over my shoulder. Tomorrow, we get to see whether it worked.