Hot start PCR is a variant of the polymerase chain reaction (PCR) developed to suppress enzymatic activity (usually Taq DNA polymerase) until the first denaturation step has been accomplished. This avoids having the PCR reaction sit at room temperature during assay setup (and prior to thermal cycling) when nonspecific amplification, a cause of PCR failure, can occur.
This Hot Start PCR video will show you how our Hot Start PCR reagents and methods accomplish supression of enzymatic activity by making modifications to the DNA polymerase, block amplification and remain inactive until higher temperatures are reached.
Learn more about our complete PCR portfolio and how to select the right reagents for your experiment.
Polymerase chain reaction (PCR) is a method for amplifying specific fragments of DNA. During assay setup, prior to thermal cycling, as PCR reactions sit at room temperature, nonspecific amplification can occur and lead to PCR failure.
To avoid nonspecific amplification, several methods known as hot start PCR have been developed that suppress PCR enzymatic activity until the first denaturation step has been accomplished.
Hot start PCR methods accomplish this by making modifications to the DNA polymerase – to block amplification and remain inactive until higher temperatures are reached. Common modifiers to the DNA polymerase include chemical, antibody or aptamer-based mechanisms.
In a PCR reaction you have your template, which contains the target sequence that you are interested in. You also need primers, dNTPs and hot-start DNA polymerase of your choosing.
A hot-start DNA polymerase will remain inactive at room temperature because a hot-start enzyme has been added. Therefore, you don’t have to worry about your amplification starting before entering the thermocycler.
Denaturation is the first step in the PCR reaction. The thermocycler heats up to roughly 95 degrees Celsius, which causes the double-stranded DNA helix to melt open into two single-stranded DNA templates. Simultaneously, the heat from this step also activates the DNA polymerase - hence, hot start.
During the annealing step, the temperature cools between 45-65 degrees Celsius and the single-stranded primers attach to the appropriate ends of the target sequence.
During the cycle, DNA polymerase attaches to the primed template and begins to incorporate complementary nucleotides.
Finally, during extension, the temperature slightly rises to 65-75 degrees celsius. DNA polymerase extends the sequence-specific primer with the incorporation of nucleotides that are complementary to the DNA template.
After repeating the denaturation, annealing and extension cycles approximately 35-40 times, replication is complete and the newly generated double-stranded DNA may be used for post-amplification analysis.
PCR reagents must provide consistent and specific, high yield amplification of your DNA. By using hot-start PCR methods your DNA amplification can benefit from: reduced non-specific amplification, increased target yield, and enhanced sensitivity.
Because hot-start methods vary on levels of fidelity, speed and convenience we recommend using a PCR selection guide and our technical support team to select what products are right for you at SigmaAldrich.com/PCR.