Nonspecific amplification is one of the major issues that can drastically impact PCR performance, resulting in one or more of: low yield of target amplicons, reduced sensitivity in detection of target amplicons, unreliable results for interpretation, and poor efficacy in downstream applications.
A common source of nonspecific amplification is the extension of misprimed sequences by DNA polymerases and the formation of primer-dimers. One workaround to help avoid nonspecific amplification is to prepare the PCR reaction mixture on ice. The colder temperature helps lower the activity of the DNA polymerase; however synthesis of undesirable products may still occur before the start of PCR. Another solution is to use a hot-start DNA polymerase. Hot-start modifications inhibit DNA polymerase's activity at room temperature, preventing spurious bands from nonspecific amplification.
What are the benefits of hot-start technology?
- Prevents extension of primers binding to template sequences with low homology (mispriming)
- Prevents extension of primers binding to each other (primer-dimer formation) during reaction setup
- Increases sensitivity and yield of the desired target fragments
- Enables PCR setup on high-throughput or automated liquid-handling platforms as reactions are stable at room temperature without compromising specificity
How does hot-start technology work?
Methods of hot-start PCR employ an enzyme modifier such as a chemical group, antibody, Affibody molecule, or aptamer. Two of the most common methods used are chemical modification and antibodies. While they all inhibit polymerase activity at room temperature, there are some key differences among them.
Hot-start technology | Benefits | Considerations |
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Chemical Polymerases are covalently linked with chemical groups to block enzyme activity at room temperature. Example: AmpliTaq Gold DNA Polymerase |
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Antibody Polymerases are bound by antibodies at their active sites to block enzyme activity at room temperature. Examples: DreamTaq Hot Start DNA Polymerase, Platinum II Taq DNA Polymerase, Platinum SuperFi II DNA Polymerase |
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Affibody molecule Polymerases are bound by Affibody molecules (alpha-helical peptides) at their active sites to block enzyme activity at room temperature. Examples: Phire Hot Start II DNA Polymerase, Phusion Plus DNA Polymerase |
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Aptamer Polymerases are bound by aptamers (oligonucleotides) at their active sites to block enzyme activity at room temperature. |
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