The polymerase chain reaction is a central technique in molecular biology. The technique was developed in 1984 by the American biochemist Kary Mullis. Nowadays, it is unlikely you will ever set foot in a molecular biology lab that does not possess a PCR machine, also referred to as a thermocycler. PCR enables very rapid multiplication of even the smallest amounts of genetic material: DNA or RNA, from a variety of sample types. It is therefore used in a variety of settings and fields of biology, from human genetics and plant biology, to microbiology or even criminal forensics.
The polymerase chain reaction & components
The PCR technique is based on a three-step cycle that is continuously repeated for a defined time. Depending on the type of sample, some may have to undergo a lysis step (breaking up the cells to expose the genetic material), such as for human or bacterial cells, viruses or parasites. This allows nucleic acids to free themselves from any associated proteins. Once the nucleic acids are free and the cell debris (such as cell wall or membrane, nuclei or remaining proteins) are removed, the sample can be used for PCR.
Figure 1. Protocol for the one-tube PCR reaction
In essence, using PCR, one can identify the presence of a specific genetic sequence and amplify it. To do so, a sequence that is specific for a certain gene or an organism should be targeted. As this technique is often used on samples from humans, animals, plants or bacterial cultures, genetic material from many other organisms or contamination may be present. However, if the chosen region is specific in a single organism, accurate detection will be possible regardless of the other material that is present.
The first step entails that the DNA strands are pulled apart, or denatured. This is done simply by exposing the genetic material to a temperature of approximately 94°C to 96°C. At this temperature, the hydrogen bonds between the molecules “melt” and separate.
Figure 2. Denaturation
The thermal cycler then lowers the temperature to approximately 55-68°C. This is where primers can bind the template strand, also called annealing (Garibyan & Avashia, 2013). These are short synthetic sequences of a few nucleotide base pairs (short genetic sequences) that are complementary to the gene of interest.
Figure 3. Annealing of the primer to the template strand
An enzyme called a DNA polymerase can then attach itself to the part where the primer is bound. When the temperature is raised to around 72°C again, the polymerase elongates the complementary strand. This means that the polymerase travels from the 3’ to the 5’ end of the template strand, catalyzing the attachment of new nucleotides (present in the reaction tube) to the strand on its way. The enzyme used for this reaction is called a Taq polymerase, which was derived from the Thermus aquaticus bacteria. This organism thrives in very high temperature environments, and therefore possesses a polymerase suitable to withstand these drastic temperature changes.
At the end of the elongated strand, the enzyme then “falls off”. As a consequence, the initial single strand of DNA now becomes a double stranded piece of DNA. After this last step, the temperature is again raised to denature the strands, initiating a new cycle in the amplification.This process will go on and on resulting in a lot of copies of the gene of interest.
Figure 4. Elongation from the 3’ to 5’ side of the template strand.
The term polymerase chain reaction (PCR) thus refers to the use of a polymerase in a successive reaction. With each cycle, the present strands are exponentially amplified, so that after a few cycles, a single strand is amplified to an amount sufficient to use for further analysis. The resulting molecules after the reaction are called the amplicon.
RT-PCR
The traditional PCR technique described above is only possible when starting with a double stranded piece of genetic material (DNA). In the case of RNA, the strands are not paired as they are single-stranded. As a result, an additional enzyme called a “reverse transcriptase” can be added to the PCR mix. This enzyme first reads the RNA to create a complementary DNA (cDNA) strand, which is a strand of DNA that is made based on an mRNA template. The rest of the reaction is similar to the one for PCR using DNA.
Figure 5. PCR is an exponential reaction, used to amplify genetic material
qPCR
Quantitative PCR (qPCR) or real-time PCR (not to confuse with reverse transcription, RT-PCR!) can also be done in the lab to quantify the initial amount of genetic material present in a sample. This can be achieved in two ways, specifically or unspecifically. In the first scenario, a non-specific dye (such as SYBR green) can be added to the mixture. As the DNA strands multiply, the fluorescent dye intercalates in the product. As a consequence, the amount of fluorescence reflects the total amount of DNA produced. For specific quantification, a probe-based real time PCR is used. To do so, a probe (small piece of genetic material) containing a fluorescent label and a quencher can be added to the PCR tube. A quencher is meant to prevent any fluorescence from being emitted, as long as the fluorescent label is in close proximity. The probes bind to the part of the DNA that is going to be copied. When the polymerase copies the DNA, the probe with the fluorescent label (fluorophore) is displaced, releasing it from the quencher [source]. These fluorophores accumulate in the reaction tube and, when excited using light, give off a signal that is representative of the amount of amplicon. The precise concentration of initial nucleic acid of interest present in the tube can thus be determined from the fluorescence.
Figure 6. Quantitative PCR: dye-based and probe-based
PCR in diagnosis
PCR can be used to detect the presence of a gene or to amplify the genetic material for further use. As a consequence, this highly versatile and largely automated technique is appreciated in clinical settings and beyond. PCR is used, for instance, for the diagnosis of many infectious diseases. Pathogens, bacteria, viruses and parasites, contain DNA or RNA. To detect a new pathogen, researchers only have to pinpoint a short sequence that is specific for this pathogen, and make the corresponding primers (Zauli, 2019).
Some pathogens can “hide” inside cells, and can therefore not always be detected by techniques such as microscopy. This can be the case for Malaria, for instance. Moreover, another diagnostic technique called “serology” detects the reaction of the body to the presence of the pathogen (antibodies), rather than detecting the genome of the pathogen directly. However, these antibodies produced to defend the organism can sometimes take a few days or weeks to develop.
In hospital settings, it is therefore preferred to take a patient’s sample and run a PCR test using primers specific for a certain disease, such as Malaria, SARS viruses or Ebola. In the case of the current pandemic, nasal swabs are taken from suspected patients and run by hundreds or thousands in labs all over the world. The accuracy and versatility of the PCR technique have made the method an indispensable “golden standard” in medical settings.
References:
Garibyan, L., & Avashia, N. (2013). Polymerase chain reaction. The Journal of investigative dermatology, 133(3), 1–4. https://doi.org/10.1038/jid.2013.1
http://www.karymullis.com/pdf/pr-1993.pdf
https://en.wikipedia.org/wiki/Polymerase_chain_reaction
https://en.wikipedia.org/wiki/TaqMan
Zauli, D. A. G. (2019). PCR and Infectious Diseases. In Perspectives on Polymerase Chain Reaction. IntechOpen.
About Violette from iGEM Leiden 2020
My name is Violette and I am part of the iGEM Leiden 2020 team, Rapidemic, for which we attempt to develop a rapid diagnostic test for infectious diseases, using a nucleic acid detection method. I am also first year master student in biomedical sciences. I have always had an interest in biology and biochemistry, as well as molecular biology. Therefore, the topic of PCR is close to both my personal interest and, of course, to the project our team at Leiden University is working on!