The Polymerase Chain Reaction (PCR)
How many times have you said to yourself, "Why didn't I think of that?" Many scientists must have had that thought when Kary Mullis described the polymerase chain reaction (PCR) in 1984. While Mullis was driving to his cabin in northern California one evening, he was thinking about a method for determining nucleotide sequences in DNA molecules. The technique used DNA replication enzymes to synthesize short strands of DNA in a test tube. After running through several scenarios in his head, he realized that he could create a chain reaction in a test tube. Every reaction would double the amount of DNA synthesized, causing an exponential growth in the number of DNA molecules created. Twenty cycles of DNA replication would create from a single molecule a million identical DNA molecules. For years scientists had worked with replication enzymes in DNA synthesis experiments, but none of them had conceived the power of a chain reaction in a test tube.
The polymerase chain reaction allows scientists to replicate (duplicate) specific DNA sequences in small test tubes. The reaction requires DNA polymerase, nucleotides (actually nucleoside triphosphates, the high-energy precursors to nucleotides), short (approximately 20 nucleotide) single-stranded DNA molecules called primers, and DNA from an organism to serve as a template. The reaction follows these steps:
1. The primer is made with the aid of a DNA (gene) synthesizer. The primer DNA sequence is critical because it determines what piece of an organism's DNA will be replicated during the PCR reaction.
2. DNA polymerase, nucleotide building blocks, primers, and DNA template are mixed together in a small test tube.
3. The test tube is placed in a thermal cycler. This machine is capable of rapidly heating and cooling samples.
4. The tube is heated to approximately 94°C to separate the template DNA into single-stranded molecules. In a living cell, enzymes carry out this process. In an experimental setting, however, heat provides a simple way to make the DNA single-stranded.
5. The tube is then cooled to 35°C-60°C. This allows the primers to locate and bind to complementary base sequences on the template DNA. Remember that DNA molecules are typically double helices with adenine-thymine pairs and guanine-cytosine pairs.
6. The tube is heated to 72°C, which is the optimal temperature for the DNA polymerase used in PCR reactions. Because the enzyme for PCR is derived from thermophilic (heat-loving) bacteria, it is not destroyed by heating (see Chapter 17). DNA polymerase locates the short, double-stranded regions where primers have bound to template DNA; it then uses nucleotide building blocks to make double-stranded DNA near the primer.
After completion of the PCR reaction, millions of copies of a double-stranded DNA molecule are present in the test tube. The real power of the reaction, though, comes from its ability to amplify only the DNA sequence near the primer binding site. This allows a researcher to make an endless supply of a specific segment of DNA. The illustration (Box Figure 13.1) shows how a target DNA sequence is amplified through cycles of PCR.
The polymerase chain reaction has become a standard tool in many research laboratories. Countless applications for this technique have been developed and more are on the way. PCR is an important tool because (1) large quantities of DNA can be synthesized from very small samples and (2) only the target DNA sequence is amplified.
How is the PCR reaction used today? Here is a short list of examples:
1. Forensics. Samples of hair, blood, and skin contain enough DNA to be amplified by PCR. A comparison of amplified DNA samples from a crime scene with DNA from a suspect can provide compelling evidence.
2. Disease detection. Primers have been developed specifically to amplify DNA from pathogens. For example, a person's blood could provide template DNA for the PCR reaction with a primer that binds only to a sequence from the human immunodeficiency virus (HIV). Then, even low levels of DNA produced by the virus would be amplified by PCR. This makes early disease detection possible. PCR is also being used to identify pathogens in plants.
3. Food safety. Pathogens in food can be detected quickly and accurately using PCR. Primers specific for pathogenic strains of Escherichia coli can detect very low levels of the pathogen in hamburger. Similar tests are available for detection of the organisms that cause Salmonella poisoning and botulism, among others.
4. Fetal testing. A small sample of fetal tissue can be amplified using primers specific to DNA regions that, in mutant form, cause human diseases. Primers are available to detect sickle cell anemia, phenylketonuria,
Stern-Jansky-Bidlack: I 13. Genetics I Text I I © The McGraw-Hill
Introductory Plant Biology, Companies, 2003
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