What is hot-start PCR?

2:25

"Here's a problem, and solution, worth knowing about. During the reaction setup for PCR, primers can bind nonspecifically to DNA templates or to each other. These misprimed DNA duplexes can be extended by the DNA polymerase. If you're using a proofreading enzyme, primers can degrade. Both scenarios can lead to nonspecific amplification, primer dimers, and reduced yield of the desired amplicon. Hot-start PCR is a simple solution. It was developed to prevent DNA polymerase activity during PCR setup. A modifier such as an antibody, affibody, chemical modification, or aptamer is used to inhibit enzyme activity at room temperature. The modifier is released during the initial heating step of PCR, or “hot start."" This results in a functional DNA polymerase. Several applications may see benefits from hot-start PCR. For example, in genotyping or sequencing where target DNA may be low, hot-start PCR helps improve PCR specificity and minimize false-positive amplification. In high-throughput PCR where assembled reactions may sit at room temperature for a while, the hot start helps prevent nonspecific amplification and primer degradation during long waits. Here are two examples of hot-start PCR enzymes. Applied Biosystems AmpliTaq Gold DNA polymerases rely on a chemical modification. On the other hand, Invitrogen Platinum DNA polymerases utilize Platinum hot-start technology with proprietary antibodies, which prevent nonspecific amplification and primer degradation. Platinum DNA polymerases are often regarded as “true” hot-start enzymes because their activity is fully inhibited until heat activation. See which hot-start DNA polymerases are right for your PCR at thermofisher.com/hot-start-pcr"

View More
View Less

Share this video

Embed