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PCR (Polymerase Chain Reaction Test) (cont.)

How is PCR (polymerase chain reaction) done?

In 1983, Kary Mullis figured out the basic steps to amplify DNA sequences. He and Michael Smith were awarded the Nobel Prize for developing this procedure in 1993. There are a few basic steps that are followed in sequence; PCR can be done in a single tube with appropriate chemicals and a specially designed heater. The reagents or chemicals needed are as follows:

  • A sample that contains a nucleotide sequence (from blood, hair, pus, skin scraping, etc.)
  • DNA primers: short single stranded DNA that attaches to nucleotide sequences that promotes synthesis of a complementary strand of nucleotides
  • DNA polymerase: an enzyme that, when the DNA has a primer bound, goes down the DNA segment attaching DNA building blocks to form complementary base pairs and thus synthesizes a complementary nucleotide strand of DNA (the introduction of a heat resistant DNA polymerase, Taq polymerase, derived from heat-resistant bacteria, markedly improved the ability to perform PCR)
  • A large excess of DNA building blocks termed nucleotides (Adenine, Thymidine, Cytosine and Guanine, abbreviated as: A, T, C and G, respectively) are present in the solution. When these blocks are linked together, they form a nucleotide sequence or a single strand of DNA. When these building blocks bind their complementary building block by weak hydrogen bonds (for example, A will only bond with T and G only with C) a complementary DNA nucleotide sequence is formed and bound to the original single stranded DNA. When the binding is completed, a complementary double strand DNA is formed in a specific sequence.

PCR, then, begins with a segment of DNA from a sample that is placed in a tube with the reagents listed above. The solution is heated to at least 94 C (201.2 F); this heat breaks the hydrogen bonds that allow complementary DNA strands to form, so only single strands exist in the mixture (this is termed denaturation of double stranded DNA).

The mixture is allowed to cool to about 54 C (129.2 F). At this temperature, the DNA primers and DNA polymerase bind to individual single stranded DNA (this is termed annealing of the DNA). Because the building blocks are in excess (high concentration) in the mixture, the polymerase uses them to make new complementary strands of DNA (termed extension of the DNA) and this process is more rapid at 72 C (161.6 F). This process creates a new double-stranded DNA molecule from each of the single strands of the original molecule.

This cycle is repeated about 40 times in a machine termed a thermal cycler that automatically repeats the heating-cooling cycles, with the amount of each DNA sequence doubling each time the heating-cooling cycle is completed. What initially was a single short segment of DNA can be amplified to about 100 billion copies after 40 doubling cycles.

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