Polymerase Chain Reaction

Last week I performed my first polymerase chain reaction (PCR) in the Moore Lab. PCR is a widely used technique to amplify a desired segment of DNA. The following is an explanation of the general process:

Preparation

To prepare a PCR, several reagents are aliquotted into a 100μL PCR tube: DNA polymerase, forward DNA primer, reverse DNA primer, template DNA, dNTP's, ultrapure H2O, and a buffer solution. DNA polymerase is an enzyme utilized during DNA replication to perform complementary base pairing. However, because the solution will later be treated with heat, mammalian DNA polymerase cannot be utilized because the enzyme will denature, therefore, an enzyme called taq polymerase is used because it originates from unicellular organisms that live in the hot springs of Yellowstone National Park. Specific forward and reverse DNA primers are used in the mixture to tell the polymerase where to start and stop copying base pairs (the forward primer on the 5'-3' strand, and the reverse primer on the 3'-5' strand). Template DNA is the original sample of DNA that needs to be amplified. dNTP's are a slew of equal ratios of freely moving nitrogenous bases for the DNA polymerase to utilize. Ultrapure H2O is the the most pure of the four levels of purified water we have in the lab, and it is free of DNAse and RNAse (agents that can denature DNA and RNA). Buffer solution is used in correspondence to the type of DNA polymerase used. For example, if taq polymerase is used, then taq buffer is used, and if high fidelity (HF) polymerase is used, then HF buffer is used. (HF polymerase is used sometimes instead of taq polymerase because if its greater capability to accurately match base pairs.)

The Process

After the correct ratios of reagents are in the PCR tube(s), the sample is placed into the PCR machine (thermocycler). (See figure 1) The thermocycler is responsible for heating and cooling the sample at precise temperatures for specific lengths of time so that the DNA is uncoiled and copied correctly. Depending on the length of the desired segment of DNA and the length of primers used, the temperatures and times can vary. The Moore lab's thermocycler has a touch screen menu where the user can edit the entire temperature sequence as needed. First, all samples begin at a temperature of 98°C for 30 seconds, then 10 seconds so that the DNA is loosened, and the heat energy can break hydrogen bonds between the complimentary nucleotides in a step called denaturing. After the strands of DNA are separated, the thermocycler will bring the sample to a new temperature for a different amount of time for a step called annealing. Annealing is the process of attachment between the primers an their complimentary segment of DNA, meaning the primers must both have time to search and find their complimentary DNA segments, and have time to completely attach themselves to that strand. The temperature and time for this step can vary because if the template DNA is more lengthy and complex, it can take more time for the primers to find their compliments, and if the primers contain a greater number of base pairs, it will take more time for them to bind. Next is elongation; the thermocycler will cool the sample to 72°C so that the DNA polymerase can attach to the primers and add base pairs along the desired strand. The amount of time provided for this step depends on the length of the DNA segment.

The thermocycler will repeat the process of denaturing, annealing, and elongating around 35 times so that a sufficient number of copies are made. After this is done, the cycle will remain at 72°C for an additional 10 minutes so that processes end and hydrogen bonds are reformed before the sample is preserved. After processes are finished, the thermocycler will cool the sample to 4°C at a time set to infinity so that the sample can be retrieved at any time and can either be utilized immediately, or stored in the -20 degree freezer.

The most important part...

Before scientists had the technology of the thermocycler, the PCR samples would have to be manually timed and moved from one water bath to the other which was, according to them, a pain in the butt. In recognition of this new technology, some scientists got together to create The PCR Song by Bio-RAD (see video).

Video 1 Figure 1