What are you going to learn?

  • What is PCR?
  • How does PCR work?
  • Why is PCR so useful?
  • What are the limitations of PCR?
  • terms: primers, Taq polymerase, thermal cyclers, buffer, denaturation, annealing, extension

PCR or polymerase chain reaction a very powerful laboratory technique that is used for copying segments of DNA. To carry out the reaction, we need several components. We need DNA polymerase, which is going to synthesize the new DNA strands. DNA polymerase needs two primers to work successfully: one primer complementary to the 5’-end of one strand and the second one to the 3’-end. We also need four deoxyribonucleoside triphosphates (dNTPs), which the DNA polymerase will use as building blocks for the newly-synthesized strands. Of course, we must have the DNA we want to replicate and finally, we need a buffer, a special solution which contains magnesium ions and other salts that create a suitable environment for the reaction.

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Components needed for PCR:
1) target DNA
2) DNA polymerase
3) dNTPs
4) primers
5) buffer
components needed for PCR

A typical PCR consists of three reactions, more specifically of a chain of three reactions, which are repeated in multiple cycles. For that reason, the technique is called polymerase chain reaction. The first step in the reaction is to separate the two strands using a process called denaturation. The DNA is heated to about 90°C, which causes the two strands to separate. During the second step called annealing, the temperature is lowered to about 40-65°C and the primers attach (anneal) to the DNA sequence we want copy. In the final step called extension, the mixture is heated again to about 70°C and DNA polymerase synthesizes the new DNA strands in the 5’-to-3’ direction. At the end of the cycle, two double-stranded DNA molecules are produced containing the target DNA sequence. Each cycle only takes a few minutes, which also explains why PCR is such a powerful method.

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PCR steps:
1) denaturation - the two strands are separated
2) annealing - primers attach to the target DNA sequence
3) extension - DNA polymerase uses the primers to synthesize new DNA strands
PCR in detail
the products of PCR after 2 cycles

As the temperatures in PCR change so quickly, a special kind of DNA polymerase is needed. We usually use Taq polymerase, which was originally isolated from a bacterium that lives in hot springs and which is therefore stable at high temperatures. What is more, special machines called thermal cyclers are used in PCR because they can very quickly change the temperatures needed for the individual reactions.

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As the temperatues in PCR need to change quickly, we use thermal cyclers and Taq polymerase.
Taq polymerase and a thermal cycler

Applications of PCR

PCR can amplify (copy) even small amounts of DNA and for that reason is very useful in forensics. As we saw during the Covid-19 pandemic, PCR can also be used to detect the presence of a virus. If the primers complementary to the viral DNA anneal and the viral DNA is replicated, the presence of the virus is confirmed. PCR can also be used in medical genetics for screening mutations.

Limitations of PCR

The first limitation of PCR is that we need to know the nucleotide sequence of the DNA segments we want to amplify so that we can construct complementary primers. As we said, PCR can copy even small amounts of DNA and for that reason, it is very important to avoid contamination in the laboratory.  Finally, PCR can only amplify short DNA sequences and is therefore not a suitable technique for amplifying large nucleotide sequences.

References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.