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Hot Start 101 Optimizing Your PCR to Avoid Non-Specific Amplification

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Hot Start 101 Optimizing Your PCR to Avoid Non-Specific Amplification Hot Start 101 Optimizing your PCR to Avoid Non-Specific Amplification The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. Challenge Accepted Optimized PCR system for the most difficult samples or DNA templates The KOD Xtreme™ Hot Start DNA Efficient and accurate amplification of long Polymerase is your enzyme of choice genomic DNA targets for the most challenging PCR situations, M1 12 34 5 M2 including crude samples, high GC content, or repeat sequences (T/A) which can inhibit or bias PCR amplification data. The most reliable enzyme for PCR from blood, Lane Samples M1 1 kb DNA ladder lysates from complex microorganisms, 1 1.3 kb β-globin target and for minimally processed animal or 2 3.6 kb β-globin target 3 8.5 kb β-globin target plant tissue, KOD Xtreme™ Hot Start 4 17.5 kb β-globin target 5 24 kb tissue plasminogen DNA polymerase and its optimized buffer activator target conditions offer remarkable properties that M2 l /HindIII DNA Markers exceed the capabilities of comparable DNA The indicated targets were amplified polymerases. from 200 ng human genomic DNA. PCR cycling parameters for 1.3 to 8.5 kb targets: 94 °C for 2 min; 30 cycles at 98 °C for 10 s, 68 °C for 1 min/ kb. PCR cycling parameters for 17.5 Order now at and 25 kb targets: 94 °C for 2 min; 5 cycles at 98 °C for 10 s, 74 °C for 1 SigmaAldrich.com/KODxtreme min/kb; 5 cycles at 98 °C for 10 s, 72 °C for 1 min/kb; 5 cycles at 98 °C for 10 s, 70 °C for 1 min/kb; 20 cycles at 98 °C for 10 s, 68 °C for 1 min/kb. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada. 2 Introduction to Hot Start PCR Polymerase chain reaction (PCR) is an established an initial heat activation step, allowing the Taq DNA method to amplify specific fragments of DNA, with polymerase to function. Since antibodies are extremely widespread utility for applications which include sensitive to heat, the activation of antibody-inhibited gene cloning, genetic fingerprinting and diagnosis of enzymes occurs within just 1 minute4. disease. In its simplest form, a PCR reaction consists Aptamer-mediated Hot Start PCR relies on the of template DNA, a set of synthetic oligonucleotide attachment of aptamers to the Taq DNA polymerase primers that flank the target sequence, a thermostable to inhibit its function. An advantage of this approach DNA polymerase (usually Taq DNA polymerase), and is that aptamers dissociate from the enzyme at a deoxynucleotide triphosphates (dNTPs) in a suitable lower temperature than heat-labile blocking groups or buffer. These components are typically combined antibodies, accelerating PCR protocols by eliminating at room temperature ahead of multiple cycles of the need for a high temperature activation step. amplification. Moreover, aptamer-based inhibition is fully reversible, A major problem with room temperature reaction meaning that Taq DNA polymerase activity is blocked at set up is that the PCR primers can anneal to non- the end of thermal cycling5. complementary DNA sequences or form primer dimers An additional Hot Start PCR method relies on the ahead of PCR cycling. This leads to non-specific use of Hot Start dNTPs. These are modified with a amplification, which can generate highly misleading thermolabile protecting group at the 3’ terminus, results. Non-specific amplification is especially which blocks their incorporation into a growing DNA problematic in situations where there is very little strand by DNA polymerase until the protecting group is template DNA, where the template is complex, or removed by heating. Although activation of Hot Start within experiments which use several primer pairs dNTPs can take up to 10 minutes, these reagents have simultaneously for multiplexing. demonstrated improved performance compared to PCR To address the problem of non-specific amplification, using standard dNTP reagents6. several methods known collectively as Hot Start PCR have evolved. These include manual Hot Start PCR, as Reasons to choose Hot Start PCR well as several approaches focused on the inhibition of Taq DNA polymerase activity during reaction set Hot Start PCR allows researchers to benefit from up. Additionally, modified dNTPs have been developed convenient, room temperature reaction setup without which lack the capacity for incorporation into a growing data quality being compromised by non-specific DNA strand until they have been heat activated. amplification. It also provides increased sensitivity and improved yields of a target DNA fragment in Hot Start PCR methods comparison to traditional PCR, making it a preferred method for research requiring an extremely high Manual Hot Start PCR involves omitting one or more degree of specificity. key components from the PCR reaction until after A further advantage of Hot Start PCR is its suitability the first denaturation step1. This prevents the DNA for high-throughput applications. Researchers polymerase from extending primers until conditions for performing Hot Start PCR can easily set up multiple primer annealing are optimal; however, the method reactions in parallel or incorporate automated liquid is time-consuming and associated with a significantly handling platforms into protocols. This improves both increased risk of contamination. Manual Hot Start PCR the quality and reproducibility of high-throughput data, is also highly prone to operator error. while saving time, resources and precious materials. A preferred approach to Hot Start PCR is to inhibit the activity of Taq DNA polymerase during the process of reaction set up. Several methods have been developed References to achieve this, including chemical modification of 1) Maximizing sensitivity and specificity of PCR by preamplification heating, the enzyme, antibody-mediated Hot Start PCR, and D’Aquila RT et al, Nucleic Acids Res. 1991 Jul 11;19(13):3749 aptamer-mediated Hot Start PCR. 2) Simplified hot start PCR, Birch DE, Nature 1996 May 30;381(6581):445-6 Chemically modified Taq DNA polymerases are 3) Advantages of a new Taq DNA polymerase in multiplex PCR and time-release supplied with heat-labile blocking groups attached to PCR, Kebelmann-Betzing C et al, Biotechniques. 1998 Jan;24(1):154-8 specific amino acid residues. These are removed by 4) TaqStart Antibody: “hot start” PCR facilitated by a neutralizing monoclonal incorporating an initial high temperature activation antibody directed against Taq DNA polymerase, Kellogg DE et al, Biotechniques. 1994 Jun;16(6):1134-7 step of up to 10 minutes into the PCR reaction, which restores the Taq DNA polymerase to full activity. 5) Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer, Lin Y and Jayasena SD, J Mol Biol. 1997 Aug 8;271(1):100-11 Following this, PCR cycling can proceed as normal2,3. 6) 3’-Protected 2’-Deoxynucleoside 5’-Triphosphates as a Novel Tool for Heat- Antibody-inhibited Taq DNA polymerases are provided Triggered Activation of PCR, Koukhareva I and Lebedev A, Anal Chem. 2009 Jun with one or several Taq-specific antibodies bound at 15;81(12):4955-62 the enzyme’s active site. As the PCR reaction begins, the antibodies denature and become detached during 3 How Hot Start PCR Works Visualize below how Hot Start PCR suppresses enzymatic activity until the first denaturation step has been accomplished by making modifications to the DNA polymerase, blocking amplification and remaining inactive until higher temperatures are reached. Watch our video, “What is Hot Start PCR?” to learn more. Just like any standard PCR, Hot Start PCR reactions include the use of the template sequence, primers, dNTPs, and During the PCR cycle, Hot Start DNA the Hot Start DNA polymerase. polymerase remains inactive at room temperature and is activated only by increasing the temperature of the reaction. The reaction temperature decreases to During extension, the reaction approximately 45-65°C during the annealing temperature rises to 65-75°C and the step, allowing primers to attach to the Hot Start DNA polymerase extends the target sequence and the incorporation of sequence-specific primer and the target complementary nucleotides. sequence. The denaturation, annealing, and extension steps are repeated approximately 35 times to produce double-stranded DNA for additional analyses. 4 Methods to Control PCR Activation Although PCR reactions have historically been set up at dNTP-mediated Hot Start PCR room temperature, this approach is highly susceptible to non-specific amplification. At room temperature, In dNTP-mediated Hot Start PCR, modified dNTPs primers can anneal to non-complementary DNA dictate when the reaction will begin. These specialized sequences or form primer dimers ahead of PCR cycling, reagents have a thermolabile protecting group at resulting in amplification of DNA sequences other than the 3’ terminus which prevents their incorporation the target of interest. Non-specific amplification often into a growing DNA strand by Taq DNA polymerase. complicates data analysis and can even lead to a failed Elongation can only occur when the protecting group is experiment. removed via an initial heat activation step of up to 10 minutes ahead of PCR cycling. Hot Start PCR techniques minimize non-specific amplification by limiting the PCR reaction prior to In many PCR applications, Hot Start dNTPs are used cycling. Established approaches to Hot Start PCR as a direct replacement for the natural dNTPs, making predominately includes three distinct methods: the process of switching from a traditional PCR protocol antibody-mediated, aptamer-mediated, or the to a Hot Start approach straightforward. While it is incorporation of specialized Hot Start dNTPs into recommended that researchers choosing to perform protocols. Because Hot Start PCR affords improved dNTP-mediated Hot Start PCR use a mix of all four specificity, sensitivity and greater target yields than modified dNTPs, it has been observed that replacement of just one or two natural dNTPs with Hot Start dNTPs more traditional PCR methods, it is now widely used to 1, 2 support many areas of research, including cancer and is sufficient to prevent nonspecific amplification .
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