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PCR Basics

Polymerase Chain Reaction (PCR) is a method of DNA replication. The method is named after DNA polymerase, an enzyme used to catalyze the process of DNA replication. Kary Mullis developed the technique in 1984 and was awarded the Nobel Prize in Chemistry for his work in 1993.

  1. Applications of PCR

    • Common applications of PCR include DNA fingerprinting (in which DNA fragments are isolated and compared against existing data), analysis of extremely small sample amounts (which enables scientists to reconstruct extinct organisms and deceased historical figures) and disease diagnosis (including viral DNA and cancer research).

    PCR Components

    • Necessary components and reagents of a PCR reaction are: genomic DNA (the template to replicate); two primers (one forward and one reverse) complimentary to the DNA template sequence; a reaction cocktail (including Taq polymerase and a buffer solution that provides a viable environment in which the reaction will occur); and the substrate for the Taq enzyme (nucleotides that will help "build" the individual strands).

    Steps of PCR

    • The steps of the reaction are: Denaturation (during which temperature is raised to separate the DNA template strands); Annealing of the primer (during which temperature is cooled to encourage free-floating primer sequences to adhere to isolated DNA template strands); and Extension (during which the Taq polymerase adheres to the primer and uses free-floating nucleotides to replicate each isolated DNA template strand). Repeating this process several times will isolate each replicated sequence from its original DNA template. Each repetition exponentially increases the amount of replicated DNA until there is a sufficient amount for the desired experiment. For optimal temperature and other conditions, see Yale University's "Designing PCR Programs" in Resources, below.

    Stages of PCR

    • The reaction can also be described in three stages: Amplification (during which DNA is exponentially replicated); Level-Off (during which the Taq polymerase loses activity); and Plateau (at which time no more product can be produced in the current reaction). For an interactive demonstration of the PCR reaction, see University of Utah's "PCR Virtual Lab" in Resources, below.

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