Polymerase Chain Reaction (PCR)
Polymerase chain reaction, or PCR, is a technique used to take a piece of DNA and make many
copies of it. This technique is very similar to the natural process which cells use to make new copies of
DNA, but it is also a little different. Scientists use an enzyme called taq polymerase which can continue to
work even at high temperatures (75 degrees C). Additionally, scientists use a machine called a thermal
cycler to control the temperatures of the reaction.
Before starting PCR, a scientist has to do some background research to find the sequence of the
DNA he or she is interested in studying. Then, the scientist will create or have someone else create
primers. Primers are small strands of DNA which match the DNA of interest. These specific sequences are
called primers because the enzyme DNA polymerase will only work correctly if there are some
nucleotides already in place. Figure 1.3 shows a diagram of the steps in PCR. After adding your sample,
primers, an enzyme called DNA polymerase and extra nucleotides, the DNA segment of interest can be
amplified (copied many times) to produce multiple copies of the desired DNA sequence.
Primer
Addition of
nucleotides
Figure 1.3. First, the double stranded DNA is dehybridized into two single strands. Next, the primer anneals
to the strands of DNA, which are called the target strands. Then, the polymerase extends the primer by
joining each nucleotide (A,C,T,G) to the end of the hybridized primer in order to extend the DNA sequence
creating a complement to the target strand. The newly made strands are then dehybridized, and the number of
strands doubles to four. Primer annealing and extension takes place again, resulting in 8 strands, then 16. After 30
cycles there are over one billion copies of the desired region.
Developed at Tufts University with funding from David R. Walt’s HHMI Professor’s Award and the Camille
and Henry Dreyfus Foundation.
The entire technique basically has three steps.
1. Denature or dehybridize DNA: Temperatures above 94 degrees C break the hydrogen bonds
between the double stranded DNA, dehybridizing it into two single strands.
2. Primer annealing: The temperature is lowered to 45-55 degrees C to allow the primers to
anneal to the single stranded DNA.
3. Primer extension: Finally, the temperature is raised to between 75-80 degrees C in order to
activate the enzyme DNA polymerase and start the process of adding nucleotides to the
primer.
A machine called a thermal cycler is used in the PCR technique. Thermal cyclers can control the
temperature of the samples that are put into the machine, and can be programmed to go to different
temperatures and “hold” them for different times, then repeat the cycle.
Putting it into context:
In this experiment, we identified the locations of the SNPs on the mitochondrial DNA sequence for
different haplogroups of interest. Then we looked up the DNA sequence immediately around the SNPs in
the scientific literature, and sent the sequences to a company which creates primers. After we received
the primers, we tested the experiment using many different saliva samples. PCR was very important in
amplifying DNA
Questions:
1. Why is it important to have a primer when doing polymerase chain reaction (PCR)?
2. What is the purpose of PCR?
3. Why is a thermal cycler used in PCR?
4. What is the role of DNA polymerase in this process?
5. What are the main steps of PCR?
Developed at Tufts University with funding from David R. Walt’s HHMI Professor’s Award and the Camille
and Henry Dreyfus Foundation.