Wednesday, April 21, 2010

My Research Explained: Polymerase Chain Reaction

In part 2 of the double whammy of My Research Explained I will explain the Polymerase Chain Reaction. The Polymerase Chain Reaction (PCR for short) is an experiment that allows for the rapid amplification of DNA. In other words I can get a poop-ton of copies of a single DNA strand in about 2 hours. This experiment has some interesting "properties" in that it is easy to perform, takes a lot of planning, may require a ton of troubleshooting, is extremely sensitive, and is difficult to explain exactly how it works. With that said, I will do my best.

When I explained the benefits of sticky ends I briefly mentioned this technique and the reason for that is because I use PCR to copy a template of DNA (from a plasmid) to make the anchor segment of the unzipping construct. In order to accomplish this I mix some components and put them in a thermal cycling machine. Sounds simple, but this technique is so sensitive that any miscalculation can result in a failed experiment. Here are the components:

  • template DNA - I use a very tiny amount of plasmid DNA and a portion of this sequence will be in the final product. Using a small amount is important because if you have too much there won't be complete products in your final sample. Hopefully this will be clear why later. The amount I use is so small that I could literally use just a few DNA molecules and still have success. All it takes is one!
  • DNA Polymerase - A polymerase is an enzyme that polymerizes free molecules into a polymer (see the correlation?). In the case of DNA those free molecules are called dNTPs or deoxyribonucleotides.
  • dNTPs - these are the four bases of DNA in their native form. A version of one of them (ATP) just so happens to be the native energy carrier of the body, cool huh?
  • Magnesium ions - these propel the polymerase
  • DNA Primers - these are short DNA strands about 20-25bp in length that get the polymerase going because it needs to start from somewhere. They serve as the ends of your final product. In a typical PCR reaction you have a forward primer and a reverse primer where one is the beginning of the DNA strand and the other is the end.
Once you mix the proper components you the set your thermal cycler and get the reaction going. The thermal cycler is a machine that cycles temperatures as many times as you want. You can use it for a variety of applications outside of PCR, but in our case we want to cycle between 3 different temperatures:
  1. You start your reaction at 95C. This is a high enough temperature to melt our double stranded DNA template into single strands but not so high that the Polymerase denatures and ceases to function.
  2. The temperature then drops to somewhere around 60C depending on your reaction. At this temp, the primers can bind to the single stranded template and the Polymerase begins doing its thang!
  3. The temperature then rises to 72C and it is here that the Polymerase really gets going. At this temp is where your DNA Polymerase is most efficient.
You cycle those three temperatures lots of times (around 30 cycles) and each cycle gives you another set of copies. You typically keep the temp at the settings above for short time intervals (around 1 min depending on the length of your desired fragment). Now just how does this copying thing work?
First you melt your template DNA as stated so you get two single strands of DNA. Next your primers bind to the template in a process called annealing. One primer binds to each strand. Now your polymerase adds the dNTPs one by one to each primer during the extension phase. It will do this until the temperature changes back to 95C. Now you have 2 sets of dsDNA, but they aren't exactly what you want because the polymerase just keeps going until the temp changes. It didn't stop at the end (where the other primer on the other strand would be).

Now the cycle begins again and your new strands melt. New primers attach to your melted DNA. One primer binds to old template DNA and another primers binds to the newly extended DNA. The polymerase once again extends on each strand. On the template strand, the same thing as above happens. But on the new strand, the polymerase will stop because the DNA doesn't go forever. It ends exactly at the end of the first primer. Then the temp raises to 95C and we are ready for the next cycle.

Now we almost have exactly what we need. After a few cycles the amount of DNA with ends is in a much larger excess than template DNA and just long copies and that means new copies will have ends. By the end of your 30 cycles you will have millions of copies of template DNA that ends exactly where your primers are giving you exactly the product you were looking for. Technically there is some DNA that you don't want, but it is in such a small amount that it is basically like a needle in a haystack. This is why if your sample gets contaminated in even the smallest amount, it could spell disaster. Any extra DNA that ends up in a sample can be amplified because if a primer even only slightly attaches the polymerase will extend and then it will copy this 30 times.

I was going to embed a video that cooly demonstrates the PCR process, but while looking for a specific video I came across this weirdness, so you all get this:

1 comment:

Dad said...

One question,
is poop-ton an official technical term?

Otherwise good write-up.

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