Polymerase Chain Reaction (PCR)

by Siyao Wang

Fun Rating: 3/5

Difficulty Rating: 2/5

What is the general purpose? 

Polymerase chain reaction (PCR) is a lab technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.

Why do we use it? 

Oftentimes researchers are interested in studying the sequence or function of a subsection of DNA. In addition, significant amounts of a sample of DNA are necessary for subsequent molecular and genetic analyses. Therefore, such studies are nearly impossible without PCR amplification.

In an academic or industry lab setting, researchers most commonly use PCR for:

• Sequencing a piece of unknown DNA
• Studying the expression levels of certain genes
• Validating the existence of a specific DNA sequence
• Amplifying a piece of DNA to make genetically-modified organisms or for other applications

Outside of a conventional lab setting, PCR can also be used for many other applications, including in forensics for genetic profiling or in a clinical setting for disease diagnosis.

How does it work? 

To conduct a PCR reaction, we’ll need a few key ingredients: DNA template, PCR primers, DNA polymerase, dNTPs nucleotides and suitable reaction conditions

What do the ingredients do?

• DNA template: this is the DNA sequence that will be amplified or copied by the reaction. 
• PCR primers: these are pairs of short synthetic DNA fragments that bind to sites flanking the target region to be amplified on the DNA template. They mark the start and end points on the DNA template so that the DNA polymerase can come in and synthesize the products in between. 
• dNTPs nucleotides: these are the building blocks of the newly synthesized DNA fragments. They consist of the 4 bases of the DNA, dATP, dTTP, dCTP, dGTP.
• DNA polymerase: this is a heat-stable protein enzyme that will synthesize the new DNA fragments using the DNA template, primers and dNTPs. Some commonly used polymerase include Taq polymerase and Q5 polymerase. They need to be heat-stable due to some high-temperature steps the PCR reaction needs to undergo. Continue reading to find out more!
• Suitable reaction conditions: conditions usually include suitable buffers, reaction temperatures and reaction cycles.

What are the reaction steps?

Figure 1: The three-step process of the polymerase chain reaction. Image created by author with BioRender.

1. Denaturing the DNA template: DNA is incredibly stable and exists in a double-helix structure with the 2 strands held together by hydrogen bonds. To amplify a piece of DNA sequence, the double-helix structure needs to be first opened up (denatured) so that the base pairs can be exposed. This step is usually done at a high temperature around 95°C. Due to the temperature requirement of this step, the DNA polymerase needs to be heat-stable so it can still function and doesn’t get destroyed by the heat.

2. Annealing primers to the template: once the double-helix DNA template is denatured into 2 single strands, temperature is lowered slightly. At this step, the primers come in and bind the start or end positions of the target regions on the single strands. Depending on the sequence and length of the primers, as well as the DNA polymerase used, the annealing temperature can vary between 50-65°C.

3. Extending the newly synthesized DNA fragments: once the primers bind the single DNA strands, it’s time to synthesize the new fragments. The DNA polymerase attaches itself to the primers which serve as guiding points. Once the polymerase binds, it starts using the dNTPs as ingredients to copy the DNA template strands to synthesize new DNA fragments.The extension temperature is usually set at 72°C for optimal extension.

4. Repeating steps 1-3: once the extension step is done, viola we have a synthesized DNA fragment of our target region! This is called a PCR product. However, doing this just once will not give us enough materials for subsequent experiments. Therefore, we’ll need to repeat steps 1-3 often for 20-40 more times (cycles) to generate enough PCR products. And remember, with every cycle complete, the product will double. At the end, we’ll end up with an exponential growth of products of around 2²⁰ to 2⁴⁰ copies.

Edited by Ellissa Z. DeFeyter and Yasemin Cole