Making A Lot Out of A Little: the Polymerase Chain Reaction

"PCR (polymerase chain reaction) is the most important new scientific technology to come along in the last hundred years."

-Mark R. Hughes, Deputy Director of the National Center for Human Genome Research, National Institutes of Health (Powledge 1998)

Hot Springs, Motorcycle Rides, and the Nobel Prize: a Background Look at the Polymerase Chain Reaction (PCR)

At first glance, it seems improbable that a stretch of California highway, a hot spring in the Yellowstone National Park, and the Nobel Prize in Chemistry have anything in common. Nevertheless, hidden behind a facade of lose connections is "an ingenious that it revolutionizes the way people ask questions" (Rhee 1998). The breakthrough development of PCR technology by scientist Kary Mullis was conceived in 1983 (Stryer 1995) as Mullis cruised the Pacific Coast Highway from San Francisco to Mendocino on his motorcycle. Ten years later, his idea won him the Nobel Prize in Chemistry (Rhee 1998).

The beauty of Mullis' idea lies in its simplicity. In essence, PCR mimics the mechanism most organisms use to copy their DNA ("Xeroxing DNA" 1992). The main obstacle of the PCR technique is its use of a wide variety of temperatures to perform DNA replication. Most naturally-occurring, temperature-dependent enzymes are not able to cope with this variety of temperatures. It is here that the organism, Thermus aquaticus and its associated, heat-stable DNA polymerase (Taq DNA polymerase) become important. This thermophilic bacterium found in hot springs in the Yellowstone National Park produces a DNA polymerase that can withstand the high temperatures used during the PCR procedure ("From Simple Ideas" 1998).

In essence, the PCR technique allows the rapid production of multiple copies of a target DNA sequence. In this sense, PCR amplifies a segment of DNA. The cyclical PCR procedure synthesizes copies of DNA very quickly as the products of one PCR cycle act as the templates for the next cycle ("From Simple Ideas" 1998). This technique is so efficient that a target sequence of DNA can be amplified a billion-fold over the course of several hours (McClean 1997).

Not only is this technique fast and efficient, it is also applicable to the work of scientists in a variety of disciplines. PCR helps researchers in fields ranging from human health (especially with respect to HIV detection), to forensic studies, to evolutionary relationships, to ecological studies and animal behavior (Powledge 1998). PCR's multitude of applications comes from its ability to amplify small fragments of DNA which are "lost" amongst the large background of the total nucleic acid ("From Simple Ideas" 1998). In fact, there is enough DNA in one-tenth of one-millionth of a liter (0.1uL) of human saliva (which contains a small number of shed epithelial cells) to use the PCR technique to distinguish a particular genetic sequence as human. Furthermore, there is enough DNA trapped in an 80 million year-old fossilized pine resin to amplify by PCR (Brown 1995). In short, very little DNA is needed to make PCR work.

"The Whole is Greater Than the Sum of the Individual Parts": the Pieces of This Ingenious Puzzle

The concept behind PCR is so simple because there are very few ingredients needed to amplify a segment of DNA:

  • Template DNA Molecule (Target Sequence)
  • Oligonucleotide Primers
  • Deoxynucleotide Triphosphates (dNTPs)
  • Heat-Stable DNA Polymerase (Taq DNA Polymerase)
  • Reaction Buffer (Containing Magnesium)
  • Thermal Cycler
  • Round and Round: the PCR Cycle

    The PCR technique works through the repetition of a three-part cycle (Figure 1) (Brown 1995, McClean 1997, Powledge 1998, Stryer 1995). Each cycle consists of the following parts:

  • Denaturation (Strand Separation)
  • Annealing (Hybridization of Primers)
  • Primer Extension (DNA Synthesis)
  • Figure 1. Each cycle of PCR consists of three steps (Denaturation, Annealing, and Primer Extension) shown here schematically. Each part of the cycle is driven by changes in temperature. This cycle is repeated between 25 and 45 times (image based on Stryer 1995).

    At the conclusion of the first cycle, both strands of the target have been replicated and can now act as templates during subsequent PCR cycles. In addition, more than just the target sequence has been replicated (Stryer 1995). Subsequent repetitions of the cycle not only increase the amount of DNA, but also refine the amount of target DNA that is being replicated. In Figure 1, for example, b-c-d-e from the first cycle serves as a template for the synthesis of b'-c'-d' during the second cycle. Similarly, a'-b'-c'-d' from the first cycle serves as the template for the synthesis of b-c-d during the second cycle. In the end, DNA consisting of the target sequence and the flanking sequences increases exponentially (2 to the n power) while the other DNA sequences (which contain more than the target sequences and flankers) increase linearly (Stryer 1995). Figure 2 shows how repeating cycles create shorter pieces of DNA more readily than the larger fragments.

    Figure 2. Multiple cycles of PCR produce many copies of the DNA target sequence and flankers while longer sequences (target sequence, flankers, and additional DNA) increase at a slow rate ("Polymerase Chain Reaction." Biotech Graphics Gallery. 1998. <> (01 February 1998)).

    A variable number of repetitions (25-45) can be executed (Podzorski et al. 1997). In the end, the specific target sequence can be amplified between one million and one-billion fold (Powledge 1998).

    For more information about PCR, go to the PCR Jump Station: "the ultimateWeb page for information and links on all aspects of the Polymerase Chain Reaction (PCR)." Here you can link to just about any information about PCR that might interest you from catalogs to protocols to general information.

    Works Cited

    Biotech. 1998. "Polymerase Chain Reaction (PCR)." Biotech Graphic Gallery <> Accessed 01 February 1998.

    Biotech. 1998. "Polymerase Chain Reaction - From Simple Ideas." Biotech Issues & Ethics. <> Accessed 01 February 1998.

    Biotech. 1992. "Polymerase Chain Reaction - Xeroxing DNA." Biotech Issues & Ethics. <> Accessed 01 February 1998.

    Brown, J.C. 1995. "What the Heck Is PCR?" <> Accessed 01 February 1998.

    McClean, P. 1997. "Polymerase Chain Reaction (or PCR)." Cloning and Molecular Analysis of Genes. <> Accessed 01 February 1998.

    Podzorski, R.P., Kukuruga, D.L., Long, M.P. 1997. "Introduction to Molecular Methodology." In: Manual of Clinical Laboratory Immunology, 5th edn. Noel R. Rose, Everly Conway de Macario, James D. Fold, H. Clifford Lane, Robert M. Nakamura, editors. Washington, D.C.: American Society for Microbiology, 77-107.

    Powledge, T.M. 1998. "The Polymerase Chain Reaction." Breakthroughs in Bioscience. <> Accessed 01 February 1998.

    Rhee, S.Y. 1998. "Kary B. Mullis (1945- )." Biotech. <> Accessed 01 February 1998.

    Stryer, L. 1995. Biochemistry, 4th edn. New York: W.H. Freeman and Company.

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