1.31: PCR

Learning Objectives

• Define the following: PCR, amplify, Taq polymerase, primer, thermal cycler, denaturation, annealing, extension.
• Give at least three applications of PCR in microbiology.
• Tell what PCR does.
• Explain how PCR works including the three steps of PCR and what happens in each of those steps.
• Successfully conduct PCR in the laboratory.
• Successfully interpret results from a PCR experiment.

 

Introduction to PCR

Polymerase chain reaction (PCR) is a technique that scientists use to amplify (make many copies of) specific DNA regions for further analysis. PCR has a huge variety of applications including:

• diagnosis of a microbial infection
• studying disease-causing organisms
• determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples
• amplifying a target region of DNA for cloning into a plasmid vector
• detecting genetic diseases
• cloning gene fragments to analyze genetic diseases
• identifying contaminant foreign DNA in a sample
• preparing DNA for sequencing
• determining paternity
• identifying the source of a DNA sample left at a crime scene
• comparing samples of ancient DNA with modern organisms

Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities. The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further analysis. One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis while at Cetus Corporation.

 

Taq Polymerase: An Enzyme Making PCR Possible

PCR is an in vitro laboratory technique (done outside of cells - in this case in a small centrifuge tube) that takes advantage of the natural process of DNA replication. Recall that DNA replication requires a DNA polymerase enzyme to build a DNA molecule complementary to a template DNA molecule. In PCR, A heat-stable DNA polymerase enzyme is used since PCR requires high temperatures to denature DNA (separate double-stranded DNA to make the DNA single-stranded). The heat-stable DNA polymerase does not denature in these conditions since it is derived from a hyperthermophilic bacterial species ("loves" hot temperatures) called Thermus aquaticus. Taq polymerase is a DNA polymerase from T. aquaticus (taking "T" from the genus and "aq" from the first two letters of the specific epithet). T. aquaticus was first isolated from a hot spring in Yellowstone National Park and thrives in very hot temperatures.

 

DNA Replication (and PCR) Require Primers

DNA replication requires the use of primers for the initiation of replication. Recall that DNA polymerases can only elongate a DNA molecule and cannot build a new strand from the start. In living cells, DNA replication uses the enzyme primase to build primers composed of RNA that DNA polymerase can elongate to build new DNA strands. In the laboratory setting, RNA is not very stable stable, and therefore, DNA primers are used for PCR. Primers not only are necessary for a DNA polymerase to elongate and produce DNA copies, primers are used in PCR to target a specific DNA sequence that we want to copy. Primer sequences are specifically designed and engineered with a specific sequence in order to target a specific DNA region. This insures that only the DNA sequence we want to copy gets copied and not other places in the DNA template molecule(s).

For example, if PCR is used for diagnosis of a microbial infection in a human, primers would be designed with sequences that match a specific region of DNA in that microbe, and does not match DNA in other microbes or in humans (human DNA will be mixed with any samples taken from a human). If the DNA is successfully copied (amplified), and matches positive controls (samples that contain the microbe the test is looking for), that microbe is causing infection in that human.

 

PCR Uses Temperature Cycles to Amplify (Copy) DNA

PCR occurs over multiple cycles. Each cycle containing three steps: denaturation, annealing, and extension. Machines called thermal cyclers are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each of the denaturation, annealing, and extension steps.

1. denaturation: First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA polymerase is required.
2. annealing: Next, the temperature is lowered to approximately 50 °C (although this can vary based on the PCR protocol and primers). This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand.
3. extension: Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of Taq polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template.

Each cycle (denaturation, annealing, and extension) doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion.

 

Figure 1: The polymerase chain reaction (PCR) is used to produce many copies of a specific sequence of DNA.

Video 1: Animation showing what occurs inside of a PCR tube during the PCR reaction. Pay careful attention to the thermometer in the top left corner since it is showing how the temperature changes created by the thermal cycler stimulates each of the PCR steps to occur.

 

Analysis of PCR Products

In order to analyze the PCR products after the DNA region of interest has been amplified, it is common for gel electrophoresis to be used to separate DNA based on size. The PCR products can then be compared with controls and other samples to make conclusions the sample. See the chapter on DNA Fingerprinting to see how gel electrophoresis works.

 

Figure 2: A gel (produced by gel electrophoresis) of samples amplified by PCR. The first lane (left) contains a DNA ladder with DNA fragments of known lengths. The DNA ladder is purchased from a supplier and contains a variety of DNA fragment lengths to enable comparison of the DNA fragment sizes of samples with unknown lengths. Notice that there is mainly one band in each of the other four wells. This is because the DNA in the other samples has been specifically targeted by PCR creating millions to trillions of DNA copies of the same section of DNA. If a sample did not contain the DNA region targeted by the primers, no band would be present.

 

Laboratory Instructions

Laboratory instructions will vary based on the protocol your instructor is following. Your instructor will provide specific instructions for this laboratory.

 

Questions

1. What does PCR stand for?
2. Give three examples of applications of PCR.
3. What does PCR do?
4. What cellular process does PCR mimic inside of a laboratory tube?
5. What is Taq polymerase and where does it come from?
6. How is Taq polymerase different from other DNA polymerases?
7. Why does PCR require a thermostable DNA polymerase?
8. Give two reasons why primers are used in PCR.
9. Explain how primers target a specific DNA sequence.
10. Why are DNA primers used in PCR when RNA primers are used inside cells?
11. What instrument is necessary for PCR and what does it do?
12. List the three steps of PCR and what happens in each step.
13. The three steps you listed in the previous question, how many times are they repeated during the PCR process?
14. How many DNA copies are produced of the primer-targeted DNA during PCR?

 

Attributions

• Analyse d'un gel d'électrophorèse-mise en évidence de l'amplification d'ADN par PCR (Jean Pierre Rullière IJPB)-2-cliche J (22824582430).jpg by INRA DIST from France is licensed under CC BY 2.0
• Biology 2e by OpenStax is licensed under CC BY 4.0
• Chapter Image: Loading PCR mixture to thermocycler machine.jpg by 26Isabella is licensed under CC BY 4.0
• Microbiology by OpenStax is licensed under CC BY 4.0