AAT Bioquest

What are the steps of PCR amplification?

Posted February 13, 2023


PCR amplification involves three major steps: 

Step 1: Denaturation

In this first step, the DNA double helix is separated by increasing the temperature of the mixture to about 94℃ for 30 seconds to 2 minutes. The high heat breaks the hydrogen bonds between the complementary DNA strands, resulting in the creation of two single-stranded DNA, which act as a template for producing new DNA strands. This process is known as denaturation.

Step 2: Annealing

In the second step, the temperature of the solution is lowered to 54-60℃ for around 20 seconds to 40 seconds. At the lower temperature, the primers bind to their complementary sequences on the template DNA to initiate polymerization. 

Primers are single-strand DNA or RNA sequences about 20 to 30 bases in length. They serve as the starting point for DNA synthesis. The two separated strands run in directions opposite to each other and are called forward primer and reverse primer.

Step 3: Extension

In the third step, the temperature is increased again to create new strands of DNA using the original strands as templates. At the higher temperature, Taq polymerase, which is a DNA polymerase enzyme, joins free DNA nucleotides together. The nucleotide sequence in the original template DNA strand determines the order in which the free nucleotides are added. 

One PCR cycle results in the creation of two double-stranded sequences of target DNA. Each contains one original strand and one newly-created strand. The cycle may be repeated multiple times to create the quantities of DNA necessary for the desired application. A billion copies can be created in only about two to three hours using PCR. 

Additional resources

Research Techniques Made Simple: Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

Helixyte™ Green *20X Aqueous PCR Solution*