Product Overview

Effective Hot Start PCR

TaqStart ® Antibody for fast, convenient hot start

• Convenient, room temperature Add TaqStart Antibody with your Ta q -derived reaction set-up DNA Polymerase during PCR set-up • Increased reaction specifi city • Use TaqStart with any full-length

Taq polymerase Begin thermal cycling • Also available in bulk quantities >70° C Polymerase activity blocked

Th e polymerase chain reaction (PCR) is Polymerase a versatile technique for exponentially activity restored amplifying small amounts of target DNA. However, it often results in the amplifi cation Figure 1. TaqStart Antibody inhibits polymerase activity before thermal cycling begins. of non-target sequences that are also present in the reaction mix. Such “background” magnesium ions (which are required by products are rarely benefi cial, and in large Hot Start PCR Methods the polymerase) from the reaction mix, amounts can decrease the yield of the and releases them when the reaction desired product and needlessly complicate Hot-start PCR methods reduce the gener- temperature is between 50–95°C (2). its analysis. Several methods, collectively ation of nonspecifi c products and primer referred to as “hot start PCR”, have been artifacts. A variety of hot start methods Chemical modifi cation of the polymerase: developed to limit the generation of exist (1), and although the specifi cs of each In this hot start method, the polymerase background products, and provide higher vary, most function by restricting the is reversibly inactivated by chemical modi- reaction specifi city and yield. availability of an essential reaction compo- fi cation. Th e enzyme is reactivated by nent until the reaction temperature is high preheating the reaction mix at 94–95°C Nonspecific enough to prevent nonspecifi c priming. for 9–12 minutes. Th is can be problematic, Amplification For example: as high temperature reactivation can result in depurination of the DNA template (3), PCR is a very sensitive technique; however, Physical removal of essential reaction prior to thermal cycling (i.e., during reaction reducing the quality of the products produced. components: Th e earliest attempts at hot In addition, the long reactivation time set up), PCR reactions are susceptible start PCR simply delayed the addition of to the nonspecifi c binding of primers increases the length of the PCR reaction the polymerase until the reaction tempera- and lowers throughput. to template DNA, contaminant DNA, ture reached 94°C, when the DNA is fully or even other primers. At suboptimal denatured. Although this method works, Reversible, ligand-mediated polymerase temperatures, low level polymerase it requires extra handling steps, which are inhibition: Th e most promising hot activity results in the extension of these inconvenient when performing multiple start methods use an oligonucleotide or misprimed sequences, and ultimately leads reactions and increase the likelihood of antibody ligand to reversibly inhibit the to the generation of nonspecifi c products sample contamination. polymerase. Ligand-mediated inhibition is and primer artifacts (e.g., primer-dimers). superior to inactivation of the polymerase Amplifi cation of nonspecifi c background Sequestration of components within by chemical modifi cation because there products reduces the yield of target DNA, the reaction: Most of the methods in this is no need for a lengthy, high temperature as nucleotides and primers that would group use heat sensitive materials, such reactivation step. Enzyme activity is restored have been used to amplify the target as wax or agarose beads, to separate an as the ligand simply dissociates or denatures sequence get used up in side reactions. essential component (e.g., Taq DNA poly- at higher temperatures. In addition, once In addition, the presence of background merase or MgCl₂) from the rest of the inhibition has been eliminated, the products hinders the isolation and subse- reaction mix. Once the reaction temperature polymerase retains most, if not all, of quent analysis of the target product. is high enough to melt the wax or agarose, its original activity. the sequestered component is released into the reaction mix, allowing PCR to proceed. Another method of sequestration involves the formation of a magnesium precipitate. Th e precipitate sequesters

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Effective Hot Start PCR…continued

Each of the ligand-mediated inhibition A Product Size Cat. No. methods diff ers as a result of the unique 120 Ab– TaqStart Antibody 200 rxns 639250 properties of the ligand involved. During 100 + Ab 500 rxns 639251 antibody-mediated hot start, the polymerase 80 is inhibited until the antibody is denatured 60 by the high temperatures in the fi rst reaction 40 cycle. Because the antibody is completely 20

DNA Synthesized (ng) DNA Synthesized Components denatured during this cycle, it no longer 0 012345 has any eff ect on the polymerase, regardless • TaqStart® Antibody Time (hr) • Dilution Buff er of the reaction temperature. M 1 2 Related Products B bp • TITANIUM™ Taq DNA Polymerase TaqStart Antibody (Cat Nos. 639208, 639209, 639242, 639210 & In order to provide our customers with 639211) the benefi ts of automatic hot start PCR, • Human Placenta QUICK-Clone™ cDNA (Cat No. 637208) most of our PCR enzyme systems are 1,353 – blended with TaqStart Antibody. TaqStart 1,078 – Notice to Purchaser Please see the Hot Start Antibody licensing is an anti-Taq monoclonal antibody that 603 – statement on page 25. inhibits the enzymatic activity of Taq and Ta q -derived DNA polymerases during room temperature reaction assembly (Figure 1). With TaqStart Antibody, you get eff ective Full polymerase activity is restored when hot start without special heating cycles, wax, the antibody is denatured during the Figure 2. TaqStart Antibody provides or expensive enzymes. TaqStart signifi cantly fi rst PCR cycle, allowing amplifi cation to automatic hot start for increased enzyme improves PCR specifi city and product yield proceed normally. Inclusion of TaqStart specifi city and product yield. Panel A. Iso- by reducing or eliminating nonspecifi c Antibody signifi cantly improves PCR thermal extension reactions were performed at 37°C for 5 hr, using single-stranded products and primer artifacts. effi ciency and specifi city by reducing or φX174 viral DNA and TITANIUM Taq DNA eliminating nonspecifi c amplifi cation and Polymerase plus (+) or minus (–) TaqStart References the formation of primer-dimers and other Antibody. TaqStart clearly inhibited DNA artifacts prior to thermal cycling (4). In synthesis (pink line). Panel B. A 1.3 kb portion 1. Martel, M. et al. (2007) In PCR: Methods Express, Eds. Hughes S. & Moody, A. (Scion addition to our enzyme blends, we also off er of the human transferrin receptor gene was amplifi ed from a mixture of human Publishing Ltd., Oxfordshire), pp 24–25. TaqStart Antibody as a stand-alone prod- placenta genomic DNA and QUICK-Clone™ 2. Barnes, W. M. & Rowlyk, K. R. (2002) U.S. uct, which allows customers to use it with cDNA. Reactions were performed using Patent No. 6403341, Magnesium precipitate the Ta q-derived DNA polymerase of their TITANIUM Taq with TaqStart Antibody hot start method for PCR. choice (i.e., native proteins, recombinant (Lane 1) or without TaqStart Antibody 3. Lindahl, T. & Nyberg, B. (1972) Biochemistry (Lane 2). As seen in Lane 1, TaqStart 11(19):3610–3618. proteins, and N-terminal deletion mutants). greatly enhanced enzyme specifi city. 4. Kellogg, D. E. et al. (1994) BioTechniques 16(6):1134–1137. Greater Specificit y and Yield TaqStart’s ability to inhibit nonspecifi c amplifi cation can also be seen in PCR To demonstrate TaqStart’s ability to reactions (Figure 2, Panel B) in which a eff ectively inhibit Ta q-derived polymerase 1.3 kb portion of the human transferrin activity, isothermal extension reactions φ receptor gene was amplifi ed from a mixture were performed on single-stranded X174 of human placenta genomic DNA and viral DNA using TITANIUM™ Taq QUICK-Clone™ cDNA. Reactions were DNA Polymerase with and without performed with TITANIUM Taq plus TaqStart Antibody (Figure 2, Panel A). TaqStart Antibody (Lane 1) or TITANIUM Without TaqStart, TITANIUM Ta q Taq alone (Lane 2). Th e reaction containing was able to synthesize over 100 ng of TaqStart Antibody (Lane 1) resulted in DNA (red line), whereas the addition of far fewer background products than the TaqStart clearly inhibited DNA synthesis reaction lacking the antibody (Lane 2), (pink line). showing that TaqStart greatly enhanced enzyme specifi city and product yield.

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