Hot Start PCR is a technique that inhibits Hot Start Taq polymerase activity or the incorporation of modified dNTPs during reaction set up until a heat activation step occurs. Hot Start PCR allows for reaction set up at room temperature without non-specific amplification and primer dimer formation. Whereas conventional PCR is often utilized to make exponential copies of your DNA target sequence without an additional temperature-sensitive reaction activation component.
PCR reactions require several critical reagents, including the template sequence for amplification, dNTPS, buffers, and Taq DNA polymerase to enzymatically facilitate the incorporation of dNTPs and ultimately create new copies of your target sequence. The basic principle of Hot Start PCR methods and reagents is that they are designed to inhibit Hot Start Taq DNA polymerase activity, or the incorporation modified dNTPs during reaction set up until a heat activation step occurs. Various methods are available to halt Hot Start polymerase activity and include, chemical modification, antibody-mediated, and aptamer-mediated technology. For additional information, view our informative Hot Start PCR video that further explains Hot Start PCR technology and how it can improve your target specificity and yield.
The benefits of Hot Start PCR include convenient room temperature set up without nonspecific amplification and the formation of primer-dimers, both of which can reduce target sequence specificity and overall yield. Alternate PCR methods, such as nested PCR or multiplex PCR, utilize different reagents and strategies to achieve specificity and high yields for one or more amplicon. Nested PCR utilizes two sets of primers, the first set flank the sequences directly outside the target sequence for the first round of amplification. The second set of primers are specific for the target sequence from the first round, which is then used as the template for the second round of PCR using the nested primers. Multiplex PCR utilizes multiple sets of highly specific primers to amplify multiple target sequences simultaneously during the same reaction. Each PCR method provides a range of benefits and should be selected based on the specific needs of the researcher.
The KOD Hot Start DNA Polymerase is a premixed complex of KOD DNA polymerase that utilizes two highly specific monoclonal antibodies to inhibit 3′→5′ exonuclease activity at room temperature. Eliminating mispriming and primer-dimer formation, KOD Hot Start DNA Polymerase is an ideal reagent for long strand amplification of genomic DNA templates (up to 12 kb), plasmid and lambda DNA templates (up to 21 kb). Utilizing an optimized enzyme to PCR amplify long and GC-rich templates, KOD Xtreme Hot Start DNA Polymerase successfully amplifies templates from crude or difficult to amplify samples, including plant tissues and mouse tail tips. Additionally, our KOD Hot Start Master Mix provides an all-in-one solution by combining KOD Hot Start DNA Polymerase, dNTPs, and optimized reaction buffer in a 2X ready-to-use mixture.
KOD Hot Start Genomic and Lambda DNA Amplification
The figure on the left demonstrates the genomic DNA amplification using KOD Hot Start DNA Polymerase of human myosin heavy chain gene (8.4 kb) and human β-globin gene (12.3 kb). The figure on the right indicates lambda DNA was amplified using appropriate primers and KOD Hot Start DNA Polymerase. M = Markers
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FastStart™ Taq DNA Polymerase is Ideal for single target Hot Start PCR amplification and multiplex applications. Additionally, the FastStart™ High Fidelity PCR System utilizes a blend of FastStart™ Taq DNA Polymerase and a chemically modified, thermostable proofreading protein. Both the FastStart™ polymerase and proofreading protein are inactive below 75 °C and activated by the removal of blocking groups after being heated to 95 °C for just two minutes.
FastStart™ High Fidelity PCR System Amplification of Human Genomic DNA
Human genomic DNA was used to amplify a 4.8 kb fragment tPA gene. The FastStart™ High Fidelity PCR System showed higher sensitivity and specificity than enzymes from two other suppliers.
Utilizing antibody-mediated inhibition, JumpStart™ Taq Polymearse is only activated after the reaction reaches 70 °C and the antibody dissociates from the enzyme. The antibody-enzyme complex allows for convenient room temperature set up with reduced non-specific amplification for standard Hot Start PCR, qPCR and multiplex PCR. Additionally, JumpStart™ Taq ReadyMix™ combines the same features of JumpStart™ enzymes with all the remaining components needed for PCR provided by the ReadyMix™ PCR reaction mix with the optional addition of an inert gel-loading dye.
Lambda Phage Amplification with JumpStart™ REDTaq® ReadyMix™ Reaction Mix
200 ng Lambda phage DNA was amplified with Sigma’s JumpStart REDTaq ReadyMix™ (odd numbered lanes) and competitor Direct Load ReadyMix (even numbered lanes).
While alternative Hot Start PCR reagents utilize antibody-mediated methods, or chemically modified DNA Taq polymerases, AptaTaq™ Fast DNA Polymerase incorporates aptamer technology to inhibit non-specific application during reaction set up at room temperature. Furthermore, AptaTaq™ Fast PCR Master conveniently provides the aptamer-mediate Hot Start Taq polymerase and all reagents, except the DNA template and primers, for fast reaction set up and even faster reaction times. Activated at temperatures above 70 °C, AptaTaq™ Fast DNA Polymerase is ideal for specific priming and fast PCR applications.
PCR Amplification with AptaTaq™ Fast PCR Master
PCR reactions using AptaTaq™ Fast PCR Master suggest superior amplification compared to competitor master mix.
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