Polymerase Chain Reaction (PCR)
PCR is a technique that allows one to make many copies of the same piece of DNA. PCR does not allow one to make many copies of the entire DNA from an organism; instead there are many copies generated of a specific piece of DNA.
This technique uses many of the steps that you have learned when you studied DNA replication.
For example, hydrogen bonds holding the two strands of DNA together are typically broken by helicase in the cell, but in PCR, high temperatures are used to break the hydrogen bonds. Both in
DNA replication and in PCR, DNA polymerase then uses free nucleotides to make new daughter strands opposite each parent strand. But in PCR, we have to use a special DNA polymerase that won’t denature in the near boiling temperatures needed to separate the DNA strands. That DNA polymerase used in PCR comes from a bacterium that normally grows in boiling hot springs! In a live nucleus there are DNA nucleotides floating around for DNA polymerase to use, so we have to add nucleotides to the PCR solution also. One big difference from DNA replication is that PCR only copies a particular region on one chromosome, not whole chromosomes. How do we choose which region to make many copies of (amplify)?
Remember, that in DNA replication, DNA polymerase has to attach to a “primer”, a short chain of nucleotides, before it can begin. We can choose which parts of the DNA we want to amplify by making primers with a base sequence that we know is complementary above and below the region we want amplified. So after the DNA strands separate, the primers attach, then the DNA polymerases attach and copy the piece of DNA between the primers, the region in which we’re interested.
So in a PCR reaction in a small tube we would have the following:
-DNA
-DNA Polymerase and a buffer that maintains the pH of the cell
-many DNA nucleotides
-many copies of 2 short DNA Primers, one for each side of the region of interest
The tube is then placed in a machine referred to as a Thermal Cycler. The machine can quickly go to preprogrammed temperatures. First, it goes to a very high temperature to break the hydrogen bonds; next it goes to a much lower temperature to allow the short DNA primers to bind at their complementary regions so that they can direct the
DNA polymerase as to where it should make new
DNA molecules; and finally, the temperature changes a third time to allow the DNA polymerase to make new DNA molecules with the nucleotides that are in the reaction. This is called a PCR cycle, and the cycle is repeated over and over again by the Thermal Cycler in order to get many copies of the region of DNA in which one is interested.
PCR cycles are not all identical. Different applications require variations in PCR cycles. PCR cycles can vary in temperatures and also in the length of time spent at any one temperature during a cycle.
After some preliminary steps, the PCR cycle that we will use is the following:
94 0 C for 60 sec - DNA Denaturation Step
55 0 C for 30 sec - Primer Annealing Step
72 0 C for 90 sec - Extension Step - New DNA molecules are made
The Thermal Cycler is programmed to do 30 of these cycles, and produces 2 30 pieces of the DNA that you are interested in.
Materials
Microbial DNA from the DNA isolation
1 PCR reaction in tubes (on ice)
Permanent markers
Equipment
Ice bucket
Micropipettor and micropipette tips
Thermal Cycler
Procedure
1. You will be supplied with a PCR tube at your station. Each tube contains the following:
PCR Buffer
Nucleotides
A pair of primers
DNA Polymerase
We need to set up the PCR reaction with our microbial DNA.
2. Adjust the micropipettor to 2. Pipette your bacterial DNA up and down several times with a slight stirring motion to mix the contents of the tube with your DNA.
3. Store your tubes on ice until everyone in the class is ready to place them in the Thermal Cycler.

4. Your instructor will help you load the tubes in the Thermal Cycler.
5. Watch the temperature and time as it goes through a cycle.
Information About Our Amplified DNA products
The primer pair that we will use to amplify our DNA of interest is a pair that is complementary to a region within the ribosomal genes.
The ribosomal gene for the small subunit ribosomal
RNA is normally located 5’ to the ribosomal genes for the large subunit of the ribosome. These genes are separated by DNA that is referred to as internal spacer region. The internal spacer region varies in length among bacteria from 300 nucleotides to
1100 nucleotides. This is the region that we will amplify. The different sized fragments will be a molecular way to assess the diversity of the microbes at our wetland site.