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

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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 . neurogenerative diseases. Supporting applications that require Antibody-mediated Hot Start PCR enhanced specificity Antibody-mediated Hot Start PCR uses Taq DNA While researchers performing any traditional PCR polymerases which are provided with one or several experiment can benefit from switching to a Hot Taq-specific antibodies bound at the enzyme’s active Start PCR approach, the technique has particular site. During an initial heat activation step ahead of PCR utility in applications which require superior cycling, the antibodies are denatured and detach from specificity and accuracy. These applications include the enzyme to restore it to full functionality. the characterization of novel mutations in cancer specimens3, multiplex PCR for infectious disease Since antibodies are extremely sensitive to heat, the diagnosis4, detection of contamination in blood activation step takes just 1 minute. This represents products5, and HLA DNA typing of forensic samples6. a significant advantage over chemically modified Hot Hot Start PCR is also relevant to microbial forensics, Start Taq DNA polymerases, where removal of heat- an emerging scientific discipline dedicated to analyzing labile blocking groups from the enzyme can take up to evidence from a bioterrorism act, biocrime, or 10 minutes. inadvertent microorganism/toxin release7. Prominent research areas have also advanced as a Aptamer-mediated Hot Start PCR result of using Hot Start PCR, including Alzheimer’s During aptamer-mediated Hot Start PCR, Taq DNA disease, rheumatoid arthritis, systemic lupus polymerases are inhibited as a result of aptamer erythematosus (SLE), HIV and amyotrophic lateral binding. Compared to antibody-mediated polymerases, sclerosis (ALS). For example, Hot Start PCR has these engineered oligonucleotides, or aptamers contributed to the finding that some genes, which are dissociate from the enzyme at a lower temperature and known to participate in amyloid-β processing, show shorten protocols by eliminating the need for a specific significant variability between individuals which may heat activation step. result in a predisposition to late-onset Alzheimer’s disease8. Additionally, the use of Hot Start PCR to An additional benefit of aptamer-mediated Hot Start analyze samples from patients with rheumatoid arthritis PCR is that enzyme inhibition is fully reversible, has indicated that endogenous nucleic acid compounds meaning that Taq DNA polymerase activity is blocked might participate in joint inflammation by activating at the end of thermal cycling. This selectivity can be immune cells within affected areas to produce pro- especially advantageous in workflows where there is inflammatory cytokines9. a significant delay between analysis of the first and last samples within a group (e.g. droplet-based digital PCR), where undesirable enzyme activity can increase the background signal in negative samples over time.

5 Hot Start PCR offers many advantages References 1) 3’-Protected 2’-deoxynucleoside 5’-triphosphates as a tool for heat-triggered Hot Start PCR techniques have been widely adopted activation of polymerase chain reaction, Koukhareva I and Lebedev A, Anal by the scientific research community for the many Chem. 2009 Jun 15;81(12):4955-62 advantages they offer over traditional PCR methods: 2) Heat activatable 3’-modified dNTPs: synthesis and application for hot start PCR, Koukhareva I et al, Nucleic Acids Symp Ser (Oxf). 2008;(52):259-60

• Minimize non-specific amplification 3) A novel p53 mutation hotspot at codon 132 (AAG-->AGG) in human renal cancer, Dahiya R et al, Biochem Mol Biol Int. 1998 Feb;44(2):407-15 • Convenient room temperature reaction setup 4) Multiplex PCR: optimization and application in diagnostic virology, Elnifro EM et • Improve sensitivity al, Clin Microbiol Rev. 2000 Oct;13(4):559-70 5) A sensitive PCR method for detecting HCV RNA in plasma pools, blood products, • Greater target yield and single donations, Saldanha J and Minor P, J Med Virol. 1994 May;43(1):72- 6

• Suitable for high-throughput applications 6) HLA typing of aortic tissues from unidentified bodies using hot start polymerase chain reaction-sequence specific primers, Sato Y et al, Leg Med (Tokyo). 2003 • Compatible with automated liquid handling platforms Mar;5 Suppl 1:S191-3 7) Public health. Building microbial forensics as a response to bioterrorism, • Enhance data reproducibility Budowle B et al, Science. 2003 Sep 26;301(5641):1852-3

8) Age-Specific Epigenetic Drift in Late-Onset Alzheimer’s Disease, Wang SC et al, High quality reagents for Hot Start PCR PLoS One. 2008 Jul 16;3(7):e2698 9) Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial fluid Using our highly optimized reagents, researchers of patients with rheumatoid arthritis, Hajizadeh S et al, Arthritis Res Ther. can enjoy the performance benefits of Hot Start PCR 2003;5(5):R234-40 and the convenience of intuitive reaction setup. As pioneers in PCR technology, Roche Hot Start PCR reagents provide improved specificity compared to competitor Hot Start PCR components, generating data of the highest quality and results that you can trust. We are committed to helping you reach your next breakthrough by offering a variety of Roche Hot Start PCR products, including antibody-mediated and aptamer-mediated Hot Start Taq DNA polymerases, and a wide range of buffers and other supporting reagents. Whichever form of Hot Start PCR you choose, we have a product to meet your requirements.

To view our complete Hot Start PCR portfolio, visit SigmaAldrich.com/hotstartpcr

6 Stop Mis-Priming New PCR Additives for Better Hot Start Reactions and Improved Specificity

Discover three unique additives from ThermaGenix to improve PCR sensitivity and product yield by preventing mis- priming. ThermaStop™, ThermaGo™, and ThermaStop™-RT reagents are easy- to-use PCR reagents that act at different temperatures to suppress errors before, during, and after amplification. • Enhance PCR specificity and sensitivity

• Make multiplexed reactions more The ThermaStop™ additive improves PCR reaction specificity accurate and yield resulting in cleaner amplification.

• Enhance the quality of downstream M = Low Range DNA Size Marker applications, including next-generation Starting Material: 60 pg Human Genomic DNA sequencing (NGS), deep sequencing, = No ThermaStop™ TS = ThermaStop™ added and Gibson Assembly

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