PCR: A simple explanation
By Mark Jackwood
Poultry Diagnostic and Research Center, Department of Population Health
University of Georgia, Athens, GA
The genome of a disease agent in a clinical sample, for the most part, cannot be directly detected because there is a very small amount of nucleic acid (DNA or RNA) present. But, using a technique to amplify the nucleic acid, one can obtain sufficient quantities that can be easily detected by a number of different methods.
Amplifying nucleic acid in a clinical sample is accomplished with PCR. The acronym PCR stands for Polymerase Chain Reaction. Polymerase is the enzyme found in cells that makes DNA. It requires a template DNA (the DNA from the organism in the sample) from which it copies a complementary strand of DNA. The new strand is complementary because in double stranded DNA the nucleotides A, C, G and T bind to each other by hydrogen bonds where A always binds with T and C always binds with G (see Figure 1). In the PCR test, the polymerase is exploited to synthesize new strands of DNA over and over again, thus creating a “chain reaction” to generate a lot of DNA very quickly.
The PCR test not only amplifies DNA to detectable levels, it can also be used to specifically identify disease agents. Since each organism has a unique set of genes, or more accurately gene sequences, a PCR test can be developed to detect a specific gene sequence, thereby identifying the organism in the sample. This is done using primers.
Before the polymerase can synthesize a complementary strand of DNA it needs a to know where to start. In the PCR test, primers tell the polymerase where to start. Primers are short (18 to 25 nucleotides) single stranded pieces of DNA. They can be synthesized in the laboratory using any combination of A, C, G and Ts. Primers bind to single stranded DNA (template) in a specific manor (A with T and C with G). The polymerase enzyme detects the presence of a primer bound to the DNA template and starts synthesizing a new strand beginning at the end of the primer. Since primers can be synthesized to any sequence desired, a primer can be created specifically to bind a unique sequence on the DNA template, thereby forcing the polymerase enzyme to synthesize a particular segment of the DNA. Furthermore, if a specific complementary sequence to the primer does not exist on the DNA template, the primer will not bind and no amplification will occur.
The polymerase enzyme can only synthesize a new strand of DNA in one direction (it can’t go backward). This characteristic is used to set up the chain reaction. In the PCR test, two primers are used. Since DNA is double stranded, one primer is designed to bind to the template strand and another primer is designed to bind to the complementary strand such that the new strand of DNA is synthesized in the direction of the other primer (see Figure 2). Synthesis in the direction of the other primer creates a strand of DNA with a complementary region to the opposite primer. Thus, in the next round of DNA synthesis, the new strand of DNA can act as template since the opposite primer can bind to the complementary region.
In the PCR test, 40 rounds or cycles of DNA synthesis are typically conducted by simply changing the temperature of the reaction. A thermocycler is used to automatically cycle the reaction between three different temperatures. First the double stranded DNA is heated to 95°C to separate the strands by breaking the hydrogen bonds holding it together. Since heating to 95°C would inactivate most polymerase enzymes, a thermostable polymerase termed Taq polymerase is used in the PCR. Next, the reaction is cooled to somewhere between 35°C and 65°C (depending on the length and sequence of the primers) to allow the primers to bind to the single stranded DNA template. Finally the reaction is warmed to approximately 72°C, which is the optimal temperature for the polymerase enzyme to synthesize a new strand of DNA. This completes one cycle. Then the process is started over again by denaturing the newly synthesized double stranded DNA at 95°C, annealing the primers (35°C-65°C) and synthesis of more DNA (~72°C). After each cycle the amount of DNA in the sample is essentially doubled. After 40 cycles, over a billion copies of DNA can be created from just one starting DNA template. And since each cycle only takes a few minutes or less, the whole reaction can be completed in 1 to 4 hours. For an animated depiction of the PCR test go to http://www.dnai.org/b/index.html and follow the Techniques, Amplifying and PCR animation links.
Reverse transcriptase-PCR or RT-PCR is identical to PCR except the starting template is RNA instead of DNA. It is necessary to use RT-PCR when detecting nucleic acid from RNA viruses like Newcastle disease virus, infectious bronchitis virus or avian influenza virus—among many others. To amplify RNA, it must first be converted to DNA. This is done with an enzyme called reverse transcriptase or RT. Reverse transcriptase is a polymerase that can use RNA as a template to synthesize a complementary strand of DNA. Once a DNA copy is created, traditional PCR can be used to amplify it.
There are a number of ways to detect amplified DNA once the PCR is completed. A common technique is visualizing the DNA after it is amplified on an agarose gel. Electrophoresis on an agarose gel separates DNA by size. The DNA is then visualized using ethidium bromide, which incorporates into the DNA and fluoresces with UV light. The amplified DNA can also be sequenced to provide even more information about the disease agent. Sequencing the PCR product is typically done when it is necessary to determine the type of pathogen present as with multiple types of infectious bronchitis virus or avian influenza virus subtypes. A variation on PCR called real time PCR or quantitative PCR can be used to detect DNA in the reaction as it is being amplified. This is done by incorporating a fluorescent marker into the reaction. The newly synthesized DNA is detected in the ongoing reaction (in real time) by measuring the amount of fluorescence in the reaction after each cycle. Real time PCR is extremely fast (approximately 20 to 30 minutes) because the DNA segment being amplified is generally short allowing for much faster synthesis reactions. In addition, real time PCR is quantitative and can be used to determine how much of the pathogen’s nucleic acid was actually present in the sample.
Because the PCR test only requires intact nucleic acid (DNA or RNA), the disease agent does not need to be alive. This is an advantage because pathogens in clinical samples can be inactivated and safely shipped and/or imported. Typically FTATM (Finders Technology Associates) cards are used to inactivate and ship clinical samples for PCR (see a review in PIP Issue 120, Sept/Oct 2011). The disadvantage of detecting only nucleic acid is that dead as well as alive organisms can be detected in a clinical sample.
However, nucleic acid does not exist outside the cell (or virus) for very long since it is relatively fragile and susceptible to nucleic acid destroying DNAses and RNAses commonly found in the environment. Thus, this drawback is not perceived as a significant problem.
In summary, PCR uses two primers to amplify a specific segment of DNA to levels where it can easily be detected. Starting with as little as one copy of DNA, more than a million copies can be generated. When the starting template is RNA (eg. RNA viruses) RT is used to convert the RNA to DNA, and then the DNA is amplified. Real time PCR (or RT-PCR) is an extremely fast method where the amplified DNA is detected during the reaction, in real time. Finally, since the primers are designed to anneal only to a specific gene sequence, PCR, RT-PCR, real time PCR, and real time RT-PCR can be designed to specifically detect the nucleic acid of a disease agent in the clinical sample.
Article from The Poultry Informed Professional, Issue 131 January/February 2014.
Published by the Department of Population Health, University of Georgia
Editor: Dr Stephen Collett, Associate Professor
Posted on August 4, 2014