PCR principal, steps, application, type, advantage, limitation

PCR is a laboratory method used to make many copies of a specific part of DNA. It is like a DNA copy machine that can produce millions or even billions of identical copies in just a few hours

Let’s imagine this:

You find a tiny drop of blood at a crime scene, or a small sample of tissue in a hospital. Inside that small sample, there’s DNA. But it’s too little to study or test. So, how do we make enough DNA to work with?

That’s where PCR (Polymerase Chain Reaction) comes in.

“PCR is a method that allows scientists to take a small amount of DNA and amplify it, creating millions or even billions of copies”.

PCR was invented in 1983 by Kary Mullis, who later won a Nobel Prize for this discovery. It became one of the most important tools in biology, medicine, and forensic science.

Components of PCR

To perform PCR, we need a few things. Think of it like baking a cake—if you don’t have the ingredients, it won’t work.

Here are the main components of PCR:

DNA Template – This is the DNA you want to copy.
Primers – These are short pieces of DNA that attach to the beginning and end of the part you want to copy. We usually use two primers (forward and reverse).
DNA Polymerase – This is an enzyme (a kind of protein) that builds the new DNA strands.
We use a special heat-resistant polymerase called Taq polymerase (from a heat-loving bacteria Thermus aquaticus).
dNTPs (Deoxynucleotide triphosphates) – These are the building blocks for new DNA (A, T, G, C).
Buffer Solution – This keeps the chemical conditions stable for the reaction.
Magnesium ions (Mg²⁺) – Help the polymerase enzyme to work properly.

PCR Steps

Now let’s see how PCR actually works. It goes through 3 main steps in cycles (usually 25–35 times). Each cycle doubles the amount of DNA.

Denaturation (94–96°C) : The double-stranded DNA is heated to break apart the two strands.
- The process starts by heating the DNA sample to a high temperature (94-96°C). This heat causes the double-stranded DNA to “unzip” and separate into two single strands. Denaturation is important because it makes the DNA accessible for the next step, where primers can bind to the strands.
- Imagine unzipping a zipper. Now we have two single strands.
Annealing (50–65°C)- The mixture is cooled so the primers can attach to the single strands of DNA.
- After denaturation, the temperature is lowered (usually to 50-65°C). This allows short DNA sequences called primers to bind, or “anneal,” to the single-stranded DNA. One primer binds to the start of the target DNA sequence, while the other binds to the end. These primers are necessary to guide the enzyme that will copy the DNA.
- Think of primers as tiny magnets that find and stick to the right spots.
Extension (72°C): The temperature is raised to let Taq polymerase do its job. It adds new DNA letters (A, T, G, C) to build the new strands.
- Once the primers have attached, the temperature is raised to about 72°C. At this optimal temperature, an enzyme called DNA polymerase starts adding complementary nucleotides (A, T, G, C) to the DNA strand. This process builds a new strand of DNA that is complementary to the original strand. The DNA polymerase “extends” the strand by adding these nucleotides, effectively duplicating the DNA segment.
- Now you have twice as much DNA as before!

These three steps make one cycle. After 30 cycles, you can get over a billion copies of your target DNA!

Applications of PCR

So, where is PCR used in real life? Let’s explore:

Medical Diagnosis
1. Detect viruses like HIV, Hepatitis, and COVID-19
1. Identify bacteria causing infections
1. Detect cancer-related genetic changes
Forensic Science
1. Solve crimes by analyzing DNA from blood, hair, or skin
1. Match suspects to crime scenes
Genetic Testing
1. Test for genetic diseases
1. Check for mutations
Research
1. Study genes and DNA sequences
1. Make DNA for cloning and experiments
Agriculture
1. Detect GMOs (Genetically Modified Organisms)
1. Identify plant diseases

Advantages of PCR

Why do scientists love PCR? Here’s why:

Quick – It gives results in a few hours.
Sensitive – Even a tiny amount of DNA is enough.
Specific – It copies only the DNA you want.
Simple – Requires only basic lab tools.
Versatility: Applicable in various fields, from medicine to environmental science
Cost-effective – Cheaper than many other DNA tests.

Limitations of PCR

But nothing is perfect. PCR has some limits too:

Contamination risk – Even a small unwanted DNA can give false results.
Taq polymerase can make mistakes – It doesn’t have a “proofreading” ability.
Requires known DNA sequence – You need to know the start and end points of the DNA to design primers.
Only small DNA pieces – PCR works best for short DNA fragments (usually under 1000 base pairs).

Can You Answer These?

So we have understood the basic concept of polymerase chain reaction (PCR). Here are some questions to check your understanding and knowledge.

What is PCR used for?
Why do we use Taq polymerase?
What are the three main steps in a PCR cycle?
Can PCR detect viruses in a person’s body?
What are the ingredients of PCR?

Try to answer them for yourself—if you can, you’ve got the basics of PCR!

References

https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
https://www.youtube.com/watch?v=KIcxzSr6IcE&t=25s
Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal. 2002;16(1):47-51. doi: 10.1002/jcla.2058. PMID: 11835531; PMCID: PMC6808141.
https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction
Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal. 2002;16(1):47-51. doi: 10.1002/jcla.2058. PMID: 11835531; PMCID: PMC6808141. https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction