A Beginner’s Guide To PCR
In the year 1985, Kary Mullis developed a technology – Polymerase chain reaction (PCR) that revolutionized the field of molecular biology (Adams, 2020, 48-53). For the invention of this futuristic technology, Kary received the Nobel Prize in Chemistry in 1993. In the modern world, PCR is of utmost importance in the field of medicine and genetics.
What is PCR?
PCR is a technique that is used in molecular biology labs. With this method, you can quantify, sequence, and copy DNA without the use of complex lab equipment. It is the most reliable approach to make use of enzymes and thermocycling to copy DNA (Redig., 2014).
What are the main ingredients of PCR?
The following ingredients are a vital part of PCR.
Polymerases are a group of enzymes that can construct new strands of DNA in the presence of template DNA and nucleotides under a favorable environment. Initially, the PCR technique was not manageable, as the denaturing of the DNA required high temperatures, and subjecting an enzyme to extreme temperatures, such as polymerase would kill it. So to combat this issue, the researchers had to introduce new batches of polymerases after every heating cycle that was challenging and time-consuming.
However, the modern PCR technique is doable and simple. The new age scientists have new extraction sources for the polymerase. The enzyme is now extracted from thermophilic bacterias. The polymerases that take part in modern PCR are usually taken from Thermus aquaticus and Pyrococcus furiosus. Polymerase extracted from Thermus aquaticus is known as Taq, while the ones that are from Pyrococcus furiosus are called Pfu. The reason why these enzymes are commercially beneficial for PCR is that they can withstand high temperatures that is a requirement for successful PCR.
Other advantages of using Taq and Pfu for carrying out PCR are:
- They speed up the reaction.
- Capable to complete long reads.
- Efficient in reading GC rich templates (Redig., 2014).
#2 Template DNA
The primers are designed according to the nature of the template DNA. All the information is read and copied from this DNA by the polymerase. The template DNA can be of three types; genomic, cDNA, and plasmid. Although the choice mainly depends on the quality count. In order to achieve accurate results, the DNA template must be pure and intact. The quantity of DNA that a specific PCR requires depends on the source. A range of 1 pg – 1 ng is enough per PCR reaction in the case of plasmid DNA. If genomic DNA is being used, around 1 ng – 1 µg is ideal (Redig., 2014).
Nucleotides are the building blocks of DNA. Since PCR is all about DNA and its manipulation, these are vital to carry out the reaction smoothly. Deoxynucleoside triphosphates (dNTPs) are used for most DNA PCRs. The labs can buy these as a mix of dGTP, dCTP, dATP, and dTTP. However, you can also purchase dNTP separately (Redig., 2014).
The short fragments that are a part of the synthesized DNA are called primers. These are the components that bind themselves to the template DNA. PCR reactions require two types of these binding components; a forward primer and a reverse primer. The forward primer is the one that designates the initiation of the PCR reaction. However, the reverse primer signals the end of the chemical activity (Redig., 2014).
The primers that facilitate PCR reactions are short pieces of single-stranded DNA. Each primer has a length that is usually around 20 nucleotides per strand. The binding point of primers is the edge of the region that is the target for copying. With complimentary base pairing, primers attach themselves to the template DNA and assist the reaction. (J.W., 2014).
The commercial polymerases that are used in the PCR reactions come with their ideal buffers. These buffers not only maintain the pH of the reaction, but they are also necessary for speeding up the activity of the polymerase. The presence of additive metals like potassium, magnesium, and DMSO in buffers also act as optimizers in different stages of PCR reaction (Redig., 2014).
What are the Steps of PCR?
When all the ingredients are assembled in one place, and the PCR reaction is good to go, all the raw materials are gathered in the PCR tube. Apart from the typical materials that are a requirement for PCR like polymerase, template DNA, nucleotides, primers, and buffers, cofactors are also added to the mix. What follows afterwards is a series of heating and cooling reactions that promote DNA synthesis. Below are the basic steps of commercial and laboratory PCR:
Step # 1 Denaturation
The mix is subjected to a temperature as high as 96℃ for the separation and denaturation of the DNA strands. Due to this step, template DNA is available for the next phase of PCR.
Step # 2 Annealing
The reaction mix is now cooled down at a temperature range of 55℃-65℃ to assist the binding step of primers to the template DNA with the help of base pairing.
Step # 3 Extension
In the last step, the temperature is raised again, so the Taq polymerases can carry out their function of extending the primers and ultimately produce new strands of DNA. The temperature in this step is usually not more than 72℃.
In a typical PCR reaction, the cycles of these three major reactions repeat 25-35 times. It takes 2-4 hours for the completion of these cycles. If the handler manages the chain reaction in an ideal way with the right quality and quantity of the ingredients, the target region can be multiplied from just one to billions in a short span of time (J.W., 2014).
Applications of PCR
The following are the applications of PCR in today’s world.
- Amplification of a single DNA sequence to millions or billions.
- Facilitate the lab testings in the field of forensic, genetics, and diagnostics.
- Detection of birth disorders.
- Diagnostics of bacterial or DNA viral diseases (Rye, 2013, 439-459).
The bottom line is the future of genetics is PCR, and there will be more advanced technologies available to make this technique advantageous.
Adams, G. (2020). A beginner’s guide to RT-PCR, qPCR, and RT-qPCR. The Biochemist, 42(3)(London), 48-53. https://portlandpress.com/biochemist/article/42/3/48/225280/A-beginner-s-guide-to-RT-PCR-qPCR-and-RT-qPCR
J.W, K. (2014, May 3rd). The Polymerase Chain Reaction (PCR) – Cloning DNA in the Test Tube. biology pages. https://www.biology-pages.info/P/PCR.html
Redig., D. J. (2014, January 27). What is PCR?- The beginner’s guide. BitesizeBio. https://bitesizebio.com/19132/pcr-basics-what-is-pcr/
Rye, C. (2013). Biotechnology. In Biology (pp. 439-459). Openstax University.