Polymerase Chain Reaction (PCR) is a method used to make millions of copies of a specific piece of DNA or RNA. Scientists use it to study genetic material in many areas of research and medicine.

This process often uses a Polymerase Chain Reaction kit, which includes all the essential components needed for the reaction, like primers, DNA polymerase, nucleotides, and buffer solution.

It works by taking a small amount of DNA or RNA and using a machine called a thermal cycler, which heats and cools the sample repeatedly to copy the DNA many times.

There are two types of DNA you might work with:

• Genomic DNA: The main DNA found in the chromosomes of cells.
• Plasmid DNA: Small, circular pieces of DNA found in bacteria that are separate from the main DNA.

Where is PCR used?

PCR has many important uses, including:

• Studying genes and DNA in research
• Copying DNA for sequencing or cloning
• Building family trees of species based on DNA (phylogeny)
• Understanding how genes work
• Diagnosing inherited diseases
• Studying very old DNA (like fossils)
• Forensic analysis (genetic fingerprinting)

The Role of DNA Polymerase in PCR

To copy DNA during PCR, we need an enzyme called DNA polymerase. The most common one used is Taq polymerase, taken from a heat-loving bacterium called Thermus aquaticus, which lives in hot springs. 

This enzyme is heat-resistant, so it works well in PCR’s high-temperature cycles.

Another enzyme, Pfu polymerase (from Pyrococcus furiosus), is more accurate than Taq but also more expensive. It’s used when high precision is needed in copying DNA.

Oligonucleotide Primers

Primers are short pieces of DNA that act as starting points for copying DNA. The enzyme DNA polymerase can only start working if it has a double-stranded section of DNA to attach to — and that’s where primers come in.

In PCR, we use two primers (each about 20–40 bases long):

• A forward primer
• A reverse primer

These primers are specially designed to match the beginning and end of the DNA segment we want to copy. 

They stick to the DNA strands using base pairing (A pairs with T, C pairs with G), and they help guide the DNA polymerase to the right place.

Primers usually have 40–60% GC content, which helps them stick better and work more efficiently. Since DNA strands run in opposite directions (one from 3′ to 5′, and the other from 5′ to 3′), two primers are needed — one for each strand.

PCR Procedure Made Simple

PCR happens in three main steps:

• Denaturation
• Annealing
• Extension

Step 1: Denaturation

• The mixture is heated to 94°C for 3–5 minutes.
• This breaks the bonds between the DNA strands and separates them into two single strands.

Step 2: Annealing

• The temperature is cooled to 50–65°C for 10–30 seconds.
• The primers attach to the single-stranded DNA.

Step 3: Extension

• The needed temperature is raised to 72°C for 5–10 minutes.
• This is the suitable temperature for Taq polymerase to work.
• It starts building new DNA strands by adding nucleotides in the 5′ to 3′ direction.

How Long Does It Take?

These three steps are repeated 30–40 times to make millions of copies of the target DNA.

• It takes about 1 minute to copy 1000 DNA bases.
• The full PCR process usually takes 3–4 hours, depending on the size of the DNA being copied.

Each new DNA strand made in one round becomes a template for the next, helping the process multiply the DNA even faster.

To Wrap Up

After PCR is finished, gel electrophoresis is used to see how much DNA was made and how big the pieces are.

This method works by placing the DNA into a soft gel and running an electric current through it. The electricity pulls the DNA pieces through the gel, and they move at different speeds depending on their size — smaller pieces move faster, larger ones move slower.

A DNA ladder (a sample with known sizes) is run alongside the DNA so you can compare and figure out the sizes of your PCR products.