Venturing PCR: principles, applications, innovations in DNA amplification for diagnostics, forensics, agriculture and beyond

Polymerase Chain Reaction (PCR) is one of the top-most innovation and technique in the field of biotechnology and has aided to the success of different biotechnological and medical research and tests. The PCR technique, developed by Kary Mullis in 1980s, permits scientists to make multiple copies of a target DNA region, simplifying the analysis, and the manipulation of genetic material. A heat-resistant enzyme called DNA polymerase is used for replicating DNA in cycles of heating and cooling.

Polymerase chain reaction is an amplification process through which a small amount of nucleic acid (DNA or RNA) can be multiplied and further tests can be performed. The machine in which the process is performed is termed as thermo-cycler and the process of PCR was first introduced by Kary Mullis, a renowned American Biochemist (Plantsurfer, 2023).

Principles and working of PCR:

Polymerase Chain Reaction (PCR) takes place in a series of 3 steps i.e.: Denaturation, Annealing, and Elongation.

Moreover, just like any recipe, there are a few components added to the sample DNA which include DNA polymerase (Taq polymerase used most commonly), buffer solutions, primers, and di-nucleotide triphosphates (dNTPs).

1.     Denaturation:

The first step of PCR is the breakage of hydrogen bonds of the double helix of each one of the DNA strands at a temperature of 95-98 degrees.

2.     Annealing:

In this step, the temperature of the thermocycler is lowered to 50-60 degrees in order for the DNA strands to gain some stability for the primers to attach to the DNA strands so that the next step i.e. elongation can take place. The temperature is lowered in this step because the primers need a stable and comparatively lower temperature to hybridize with the DNA strands.

3.     Elongation:

This step depends on DNA polymerase and requires a temperature set between 72-78 degrees.

What happens in this step is that the DNA polymerase attaches to the DNA strands at the points where primers are already attached and then initiates the formation of complete DNA strands by initiating the attachment of dNTPs to the ends of primers.

After the respective DNA strands form double strands, they intertwine into a helix once more and the quantity of DNA strands is doubled. One PCR cycle takes up to 1-2 minutes to take place and usually more than 45 cycles take place for tests to be carried out.

Advancements in the PCR techniques:

With time and increase in technology, many advancements and modifications have been made to the PCR.

The most common advancement techniques of PCR include:

1.     Isothermal amplification:

As the name suggests, the PCR in this technique does not require the constant change of temperatures instead the amplification steps take place at  a set temperature.

This PCR technique is used in Loop-Mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA).

2.     Next-generation PCR:

This PCR technique is the most modern PCR technique and also the most accurate, sensitive, and cost-saving PCR technique.

NG-PCR takes place in two ways i.e. Multiplex PCR and digital PCR.

3.     High throughput PCR system:

The high throughput PCR technique can simultaneously amplify a large number of DNA samples and therefore is pretty convenient and user-friendly.

Real-time PCR, PCR Robots, and Microfluidic high throughput PCR are a few of the high throughput systems.

How does PCR help in diagnosis?

PCR being one of the most crucial tests in medical fields plays an essential role in pathogen identification, cancer diagnostics, transplantation screening, and mutation.

1.     Role in infectious disease identification:

A few years ago, at the onset of the pandemic especially, the importance of PCR was the most evident. With the help of PCR, the presence of bacteria and viruses can be identified in patients. Moreover, the quantity of DNA from the PCR can also determine the seriousness of the disease and how it affects the patients.

This can be done through bio typing, a method by which specific DNA sequence of the microbial species is matched with the amplified DNA and species are identified.

2.     Forensics:

PCR is one of the tests conducted alongside multiple other tests to know the cause of death. It is done to identify the murderer through their fingerprints or any other sample left behind as well as necessary for the identification of the victim’s body in any disaster or accident (Cheriyedath, 2018).

3.     Cancer diagnosis:

Apart from the above medical applications, PCR is also done in order to identify any type of mutation or abnormal growth of the patient’s DNA. It can even help to identify the type of Cancer that a person has (Polymerase Chain Reaction, 2008).

Furthermore, medical practitioners also use PCR to check the progress of the patient to the treatments he is provided with, and can also be used to detect whether the patient is completely cancer free or not.

What role does PCR play in agriculture and food industry?

As much as PCR is important in medicine, it also plays a big role in agricultural and food industry’s advancement.

1.     Crop safety:

PCR can be used in agriculture to detect the DNA of any pathogen which is a threat to the crops. In addition to this, individuals can identify pests and microbes that can infest the crop and eradicate them from that area.

2.     GMO labeling:

The GMO present in a crop can be quantified and labeled with the help of PCR technique and an analysis can be done using PCR-based assays for further use by consumers (2014).

3.     Food safety and quality control:

In the food industry, before packaging edible items, PCR-based tests are performed to identify any foreign body or contamination present in the food.

Moreover, the ingredients of the foodstuff can be traced down and noted for the presence of allergens which is written down on the package for the consumer’s knowledge (Ayu, 2021).

Environmental applications of PCR:          

Just like agriculture and diagnostics, PCR is also one of the tests very important for environmental studies (Cheriyedath, 2018).

1.     Microbial detection:

PCR can be used for the identification of different microbial organisms present in different ecosystems as well as in meta-genomics. It can also be used for the detection of pathogens that are airborne and waterborne in nature.

2.     Bioremediation:

Amplification of DNA can help detect organisms that play a role in bioremediation and the environment they are in.

For example, Humus-producing bacteria identified through PCR can be used for degrading organic waste and oil spillage in the oceans and also for monitoring oil-degrading microbes.

Conclusion:

The amplification of DNA using PCR technique is one of the biggest successes and innovation of biotechnology which has numerous applications to it. PCR-based tests can be carried out in every field of biology where the presence of microbes or identification of a DNA sequence is present. With the advancement of time, the application and techniques of PCR are increasing and so are the challenges and potential for advancements unlocking with it.

References

(n.d.). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263587/#:~:text=Isothermal%20methods%20differ%20from%20PCR,in%20less%20than%20an%20hour.

Ayu, D. (2021, January 14). PCR for Food Industry – Fast Results for microbiological testing. Retrieved from https://baliseafoodlab.com/pcr-for-food-industry/

Cheriyedath, S. (2018, August 23). Retrieved from https://www.news-medical.net/life-sciences/Polymerase-Chain-Reaction-Applications.aspx#:~:text=PCR%20is%20a%20highly%20valuable,greatly%20impacts%20public%20health%20monitoring.

Jain, H. (2014). Retrieved from https://www.slideshare.net/harshitajiancdfst/application-of-pcr-in-agriculture

Laura Maria Zanoli1 and Giuseppe Spoto1, 2. (n.d.). Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263587/#:~:text=Isothermal%20methods%20differ%20from%20PCR,in%20less%20than%20an%20hour.

PCR. (n.d.). Retrieved from National Centre of Biotechnology information: https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/#:~:text=PCR%20(Polymerase%20Chain%20Reaction)&text=PCR%20is%20based%20on%20using,can%20add%20the%20first%20nucleotide.

Plantsurfer. (2023, July 21). Retrieved from Wikipedia: https://en.wikipedia.org/wiki/Polymerase_chain_reaction

Polymerase Chain Reaction. (2008, February 7). Retrieved from Virtual Medical Centre: https://www.myvmc.com/investigations/pcr-polymerase-chain-reaction/#:~:text=PCR%20is%20extensively%20used%20in,fever)%2C%20malaria%20and%20anthrax.

 

By: Fatima Hasnain and Uzma Raza