PCR- a DNA copy machine  

Did you know that DNA can be photocopied? 

In schools and offices, copy machines are very helpful since they can quickly duplicate pages from all types of sources. Likewise, biologists often need to make a large number of copies of genetic material. They use a technology called PCR (Polymerase Chain Reaction). 

Kary Mullis, an American scientist, invented this procedure in his research in 1983 that was one of the greatest significant achievements in molecular biology. PCR revolutionized DNA research to the point that its inventor received the Nobel prize in Chemistry in 1993.

In biological laboratories, PCR is a straightforward, quick, and low-cost technology. Because DNA is unique to every living thing, scientists may frequently extract small segments of DNA of interest from a sample. Scientists then manufacture a modest number of copies to be effective, thus they will employ PCR to generate a sufficient number of copies to begin experimenting with. This method will ultimately generate a billion or more copies in a matter of a few hours. As a result, it is among the most often used techniques in genetic laboratories throughout the world, making it valuable in a multitude of studies, including the detection of hereditary disorders, the identification of genetic fingerprints, the diagnosis of infectious diseases, and plant breeding.

Components of PCR

Here are the most important components needed to make multiple copies of DNA in PCR:

DNA template or Sample

DNA sample or DNA template is the particular DNA that you want to copy. 

Primers

Primers are short single-stranded DNA fragments having complementary sequences to the template. Complementary sequence means they pair with the opposite nucleotides of the DNA strand. For example, if DNA template has nucleotide A (Adenine) then the primer strand will have nucleotide T (Thymine) to pair with it. Similarly, G (Guanine) pairs with C (Cytosine). 

Deoxynucleotide triphosphates (dNTPs)

The nucleotides (A, T, C, and G) serve as the building blocks of DNA and are needed to make new copies of DNA. 

Taq DNA polymerase

The DNA polymerase is an enzyme which attaches to the DNA template and makes new strands of DNA by adding complementary nucleotides (A,T,G, and C). Enzymes are important proteins made by the cells of plants and animals. They are catalysts and their job is to speed up the chemical reactions. Taq polymerase is a heat-stable enzyme which comes from the bacterium Thermus aquaticus.  

PCR buffer

This provides a suitable chemical environment for the DNA-Polymerase to work. 

PCR Tube

These are small tubes designed to process the PCR. All the components are mixed in a PCR tube and put into a thermocycler for making multiple copies of DNA. 

Thermocycler

It is a device that can rapidly (in seconds), and precisely, raise or reduce the temperature of the samples stored in it. 

Stages of PCR

In the laboratory, scientists copy DNA. PCR allows scientists to choose which parts of DNA they want to make billions of copies. Let us perform a quick PCR here:

Fill a PCR tube with all of the PCR components (DNA template, Primers, Taq DNA polymerase, PCR buffer, and DNTP’s). Place the tube in an automated thermocycler, which can quickly heat and cool the tube containing PCR components. 

A PCR consists of three basic stages that are repeated 30 to 40 times. Taq DNA polymerase is normally denatured after 30-35 cycles because each denaturation temperature (94°C- 96°C) for a minute affects the protein function,  and it cannot withstand it beyond 35 cycles. A billion or more copies can be obtained in as little as 2-3 hours.

1. Denaturation: The double-stranded DNA template is heated to roughly 94°C - 96°C in this stage. At this temperature, the hydrogen bonds that keep the two strands of template DNA break and split. 

2. Annealing: After denaturation, the thermocycler reduces the temperature of the PCR tube contents and prepares for annealing. In this step, the primers (yellow in color in the above diagram) bind to the complementary sequence of the denatured template DNA at around 55°C - 70°C. A wrong temperature during the annealing step can result in primers not binding to the DNA strand at all. 

3. Extension: Following the binding of primers, the thermocycler raises the temperature of the PCR tube content and prepares for extension. Taq DNA polymerase (the green bean-shape in the picture above) connects to the primers and creates a new copy of the DNA using the template DNA. Taq polymerase performs excellently at a temperature over 70°C. As a result, the extension step is often performed at 72°C. 

PCR – a plant breeding tool

In this age of industrialization, all nations rely on agriculture as their primary source of economic growth. PCR is employed in agriculture in a variety of methods, including:

• One of the goals of this technology is to identify the presence or absence of genetically modified material in a plant product. Genetically modified plants are those whose genes are altered by adding a new gene ( the new gene instructs the cell to create proteins which may determine a trait/characteristic such as flower color in plants.) using genetic engineering techniques such as CRISPR. For example, golden rice. 

• The approach has shown to be a powerful tool for the detection and diagnosis of crop diseases, and it has aided in the plant disease management. For example, there is a severe threat of infections in banana plants, which has harmed the banana production worldwide. The PCR technology was used to identify and treat various disorders. 

Conclusion

To summarize, PCR is a highly useful method for replicating DNA and diagnosing certain diseases. It is a reliable method of testing and analyzing DNA that is also quite efficient because it just takes a few hours. 

PCR is utilized in agricultural product development. This may include procedures such as, improving seed quality, gene discovery, and so on. 

In general, PCR is an extremely valuable method for genetics research. And who knows what may occur? Perhaps you will discover a new use for this DNA replicating machine in the future. 

For more interesting articles on different aspects of molecular biology, keep checking the space.

By Priyanka Biala | 19th October 2022

Priyanka is based in Toronto, Canada, with her partner and two adorable kids (a dinosaur lover and a bibliophile). Priyanka is a scientific enthusiast with a master’s in biotechnology. She has spent the last decade exploring science and technology with various research institutes and multinational corporations. She fervently setups the labs and scientific procedures, as well as participates in biotech agricultural research on a variety of crops with renowned scientists in the area. Right now, she is deep diving into the regulatory processes in the pharmaceutical industry.

References

• https://www.intechopen.com/chapters/70299
• Explainer: How PCR works | Science News for Students 
• What is PCR (polymerase chain reaction)? | Facts | yourgenome.org 
• https://passel2.unl.edu/view/lesson/d81d0eedbada/9
• Sciencemadness Discussion Board - Complete DNA Synthesis for Viral Vector - Powered by XMB 1.9.11