Premium PCR Enzymes Takara Ex Taq ™ DNA Polymerase Applications • High sensitivity, high yield PCR, even with DNA λ DNA ≤30 kb from problem organisms and samples Genomic DNA ≤20 kb Accuracy vs. Taq 4.5X • PCR requiring less optimization and increased Hot-start version √ reproducibility 3'-A ends √ • Standard to long DNA amplifications Optimized Buffer √ Premix available √ • Specific, sensitive real-time PCR with a wide dynamic range Description Takara’s Ex Taq™ DNA Polymerase combines the proven perform- ance of Taq DNA Polymerase with an efficient 3’ → 5’ exonuclease Call or v activity for unsurpassed PCR performance. In routine PCR applica- website isit our tions, the Ex Taq™ Enzyme Buffer System results in higher yields, for a lower error rates (approximately 4.5X lower, as determined by the Sample Free Kunkel method), and more reproducible results than standard Taq ! DNA Polymerase. This system also allows amplification of longer products than Taq DNA Polymerase, with 20 kb lengths possible from genomic DNA and up to 30 kb possible from λ DNA. PCR products generated with Ex Taq™ contain a mixture of 3′-A overhangs and blunt ends. Generally, >80% cloning efficiency is achieved into T-vectors. The high performance of Ex Taq™ makes it well suited for many applications. Standard, Hot-start, Premix, and Real-Time PCR (qPCR) versions are available. Robust Amplification Ex Taq™: High Sensitivity and High Yield Ex Taq: DNA Amplification from Problem Organisms and Sources Takara’s Ex Taq™ DNA Polymerase’s proven performance makes it an excellent candidate for routine PCR. The robust Ex Taq™ enzyme-buffer system results in high product yields from even very small amounts of starting DNA (0.025 pg). This system has also allowed researchers to amplify DNA from problem organisms and sources, including high polyfaccharide plants, algae, and human biopsy and fecal specimens. ■ This figure illustrates amplification of a 6 kb target from E. coli genomic DNA with Takara Taq and Ex Taq™ DNA Polymerases. PCR was performed using the indicated amounts of template, with either Takara Taq™ (Lanes T1-T4) or Ex Taq™ (Lanes E1-E4). Each 50 µL reaction contained 0.9 units of enzyme. Lanes M contain λ Hind III Amplification of a 6 kb target from E. coli DNA Markers. The Ex Taq™ lanes show superior amplification over genomic DNA with Takara Taq™ and Taq DNA Polymerase. Ex Taq™ DNA Polymerases. 2 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com Increased Reproducibility and Minimal Optimization in Standard PCR Ex Taq™ and Ex Taq™ Hot Start: Reliable, Consistent PCR Takara's Ex Taq™ DNA Polymerase and Ex Taq™ Hot Start DNA Polymerase include dynamic enzyme-buffer systems which result in reliable, consistent PCR performance for even challenging samples, and often with less time spent on optimization. The system is so robust that, in many applications, the amount of Ex Taq™ and Ex Taq™ Hot Start required is less than one-half of the enzyme amount necessary with conventional Taq DNA Polymerase 1 2 3 4 5 6 7 8 9 10 11 12 ■ This figure illustrates amplification of a 1.1 kb Bacillus sp. genomic DNA target using Ex Taq™ Hot Start DNA Polymerase vs. several competing enzymes. Lanes 1 and 2, Ex Taq™ Hot Start with 10X Ex Taq™ buffer; lanes 3 and 4, AmpliTaq Gold® with supplied buffer; lanes 5 and 6, AmpliTaq Gold® with 10X AmpliTaq Gold® Buffer; lanes 7 and 8, Advantage™ 2 Polymerase; lanes 9 and 10, Platinum™ Taq DNA Polymerase; and lanes 11 and 12, Proof-Start™ DNA Polymerase. This experi- ment illustrates improved signal intensity and lower background with Ex Taq™ Hot Start over competing Amplification of a 1.1 kb Bacillus sp. genomic DNA target. products. Specific, Sensitive Real-Time PCR (qPCR) SYBR® Premix Ex Taq™: Superior Specificity and Increased Amplification Efficiency for qPCR Takara has a wide range of real-time PCR products, including the CycleavePCR Core Kit, Ex Taq™ R-PCR Version 2.1, and the Real Time One Step RNA PCR Kit. All of these products contain Ex Taq™ Hot Start DNA Polymerase, dNTPs, Mg2+, and a newly formulated real time buffer which provides superior specificity and increased amplification efficiency in real time PCR. Takara’s newest qPCR addition, SYBR® Premix Ex Taq™ (Perfect Real Time), is a convenient (2X) premix which contains SYBR® Green I, and a separate tube of ROX reference dye. It is compatible with all major qPCR instruments, including the Smart Cycler®, LightCycler®, ABI 7000,7300, 7500 and 7700, RotorGene™, as well as other real time instruments. It can detect as few as 100 copies of starting template, and possesses a dynamic range of 7-8 orders of magnitude using a λ DNA template. Excellent standard curves for various real time instruments have been established with this enzyme. SYBR® Premix Ex Taq™ can be used for rapid reactions because it provides conditions favorable for short denaturation steps and is suitable for shuttle PCR. Amplification curve and Melting curve for ■ This figure shows amplification and melting curves using SYBR® SYBR Premix Ex Taq™ (Perfect Real Time) Premix Ex Taq™ (Perfect Real Time) on a Cepheid Smart Cycler®. using the Smart Cycler® (Cepheid) The specificity and sensitivity of this system is illustrated by the wide dynamic range and sharp melting curve. 3 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com Guide to Takara P Product Size Product size Polymerase* Amplification λ DNA Human Genomic DNA Fidelity** Proofreading Specificity Efficiency Recommended/Max Recommended/Max Activity Ex Taq™ ++++ 20 kb/30 kb 10 kb/20 kb 4.5 X Taq Yes ++ Premix Ex Taq™ ++++ 20 kb/30 kb 10 kb/20 kb 4.5 X Taq Yes ++ Ex Taq™ HS ++++ 20 kb/30 kb 10 kb/20 kb 4.5 X Taq Yes ++++ Ex Taq™ R-PCR ++++ _ _ 4.5 X Taq Yes ++++ Premix Ex Taq™ (Perfect Real Time) ++++ _ _ 4.5 X Taq Yes ++++ SYBR Premix Ex Taq™ (Perfect Real Time) ++++ _ _ 4.5 X Taq Yes ++++ LA Taq™ +++ 35 kb/48 kb 20 kb/30 kb 6.5 X Taq Yes ++ LA Taq™ w/GC Buffer +++ 35 kb/48 kb§ (20 kb/30 kb)§ (6.5 X Taq)‡ Yes ++ LA PCR Kit +++ 35 kb/48 kb 20 kb/30 kb 6.5 X Taq Yes ++ One-Shot LA PCR Mix +++ 35 kb/48 kb 20 kb/30 kb 6.5 X Taq Yes ++ LA Taq™ HS +++ 35 kb/48 kb 20 kb/30 kb 6.5 X Taq Yes ++++ Z-Taq™ +++ 20 kb/30 kb 10 kb/20 kb 3 X Taq Yes ++ Taq ++ 6 kb/12 kb 2 kb/4 kb 1 X Taq No ++ Premix Taq ++ 6 kb/12 kb 2 kb/4 kb 1 X Taq No + Taq HS ++ 6 kb/12 kb 2 kb/4 kb 1 X Taq No ++++ All of Takara’s premium PCR enzymes utilize Long and Taq Polymerase (determined by the Kunkel method), and Accurate (LA) PCR technology, which uses a mixture of the perfected enzyme-buffer system often results in less Taq Polymerase and a proofreading polymerase to gener- time spent on optimization than other long PCR polymeras- ate products with improved fidelity and extended length. By es. Standard, Hot-Start, Premix, and GC-Rich Versions of additionally optimizing buffer and reaction conditions, LA Taq™ are available. Takara has produced a line of high-performance poly- Both Ex Taq™ DNA Polymerase and LA Taq™ DNA merases and products for specialized applications. Polymerase offer excellent performance with DNA templates Takara Ex Taq™ DNA Polymerase is optimized for maximal > 5 kb in length. Ex Taq™ can amplify fragments up to 20 kb performance in both routine and challenging PCR. It offers using a genomic DNA template and up to 30 kb with λ DNA. high sensitivity, increased product yield and length, and When amplifying products from difficult templates, such as improved reproducibility and fidelity over Taq Polymerase. plant samples or “dirty” specimens, Ex Taq™ is generally the Standard, Hot-Start, Premix, and Real-Time PCR (qPCR) enzyme of choice. LA Taq™ is the best choice when working versions of Ex Taq™ are available. with very long templates (>15 kb), or templates with second- ary structure or high GC content. Takara’s LA Taq™ DNA Polymerase is optimized specifically for long PCR, and can be used to synthesize products up to Both Ex Taq™ DNA Polymerase and LA Taq™ DNA 48 kb in length. Fidelity with LA Taq™ is 6.5X better than Polymerase are available in Premix versions, which consist 4 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com PCR Polymerases Guidelines for Terminal Convenience GC-Rich Hot-Start PCR Real Time PCR Low DNA Processing Length of Transferase Activity Templates (QPCR) Enzyme Speed Primers (3’-A overhang) ++ + _ _ ≤ 10 fg 1-2 kb/min 20-30 bp Yes ++++ + _ _ ≤ 10 fg 1-2 kb/min 20-30 bp Yes ++ + ++++ _ ≤ 10 fg 1-2 kb/min 20-30 bp Yes ++ + ++++ ++++ ≤ 10 fg _ _ Yes ++++ + ++++ ++++ ≤ 10 fg _ 17-25 bp Yes ++++ + ++++ ++++ ≤ 10 fg _ 17-25 bp Yes ++ + _ _ ≤ 10 fg 1-2 kb/min 25-35 bp Yes+ ++ ++++ _ _ ≤ 10 fg 1-2 kb/min 25-35 bp Yes+ ++ ++++ _ _ ≤ 10 fg 1-2 kb/min 25-35 bp Yes+ ++++ + _ _ ≤ 10 fg 1-2 kb/min 25-35 bp Yes+ ++ + ++++ _ ≤ 10 fg 1-2 kb/min 25-35 bp Yes+ ++ ++ _ _ ≤ 10 fg 4-5 kb/min _ Yes ++ + _ _ ≤ 10 fg 1 kb/min _ Yes ++++ + _ _ ≤ 10 fg 1 kb/min _ Yes ++ + ++++ _ ≤ 10 fg 1 kb/min _ Yes of a single solution containing enzyme, buffer, Mg2+, and with separate components, or as Premix versions both dNTPs, and provide the convenience of simple reaction with and without SYBR Green I. Takara’s latest addition to assembly. Thus, the number of pipetting steps is reduced, the qPCR product line, SYBR Premix Ex Taq™ (Perfect saving time and labor, and limiting opportunities for con- Real Time), additionally includes a separate tube of ROX tamination. reference dye. Hot-start versions of Ex Taq™ DNA Polymerase and LA Taq™ DNA Polymerase, which contain a monoclonal Taq * All of Takara’s PCR polymerases are provided with dNTPs antibody, are also available. These hot-start version and buffer. enzymes provide increased specificity and sensitivity, + T-vector cloning efficiency diminishes as the length of the reduce background, and allow room-temperature reaction PCR product to be cloned increases above 5 kb. assembly. § When used with GC Buffer I. ‡ When amplifying GC-rich templates, the fidelity is reduced. Real-Time PCR (qPCR) versions of Ex Taq™ DNA ** All fidelity determined by using the Kunkel method. Polymerase are also available for high performance qPCR. Ex Taq™ Hot-Start Polymerase is supplied in Takara Bio owns the worldwide rights to LA PCR technology, and also pro- these versions with optimized high-specificity qPCR vides a wide selection of PCR Kits, including kits for RT-PCR, RACE, Competitive PCR, PCR cloning, PCR mutagenesis, and buffers. Takara’s real time products are available as Kits organism and gene screening. 5 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com Takara LA Taq ™ DNA Polymerase Applications λ DNA ≤48 kb • Long PCR (up to 48 kb) Genomic DNA ≤30 kb • PCR amplification of difficult templates due to Accuracy vs. Taq 6.5X secondary structure or high GC content 3'-A ends √ Hot-start version √ • Increased specificity and reduced background using Optimized Buffer √ Hot-Start technology Premix available √ G-Rich Version Description available √ Takara’s LA Taq™ DNA Polymerase is composed of a mixture of Taq Polymerase with a proofreading polymerase which, when used with Takara’s specially optimized LA Buffer II, provides PCR products of maximum length (≤ 30 kb for genomic DNA, ≤ 48 kb for λ DNA) with sig- nificantly improved fidelity. Because of the presence of the proofreading Call or polymerase, the fidelity of LA Taq™ is 6.5X better than Taq Polymerase website visit our alone (determined by the Kunkel Method). Furthermore, many users to note that this high-performance system requires less optimization than trial siz order your e of LA other long DNA polymerases. LA Taq™ DNA Polymerase has been Taq used by researchers with a variety of templates including smooth mus- cle phosphoprotein, Ascidians (sea squirts), Drosophila, Arabidopsis, C. elegans, and the root parasite, Orobanche cumana. For templates with high secondary structure or high GC content, LA Taq™ DNA Polymerase with GC Buffers provides two different buffers which can be used to destabilize problem hairpin loops and synthesize templates with up to 72% GC content. PCR products generated with LA Taq™ contain a mixture of 3’-A over- hang and blunt ends. Generally, >80% cloning efficiency is achieved into T-vectors. Standard, GC-Rich, Premix, Hot-Start, and Kit versions of LA Taq™ are available. Long Amplification and Excellent Fidelity LA Taq™: Amplification of 5-15 kb fragments; extensions to 48 kb possible LA Taq™: Long PCR from limited amounts of starting template Takara’s LA Taq™ DNA Polymerase overcomes traditional problems asso- ciated with efficient amplification of DNA fragments ≥5 kb through an opti- mized enzyme-buffer system. This system incorporates LA (long and accu- rate) PCR technology, and results in increased product fidelity, sensitivity, and length. With LA Taq™, routine PCR amplification of templates ranging from 5-20 kb can be expected, with lengths of up to 48 kb possible on some templates. This reliable system also allows amplification from small- er starting template amounts than other long PCR systems, making it the enzyme of choice for synthesis of long genomic DNA fragments. ■ The sensitivity of LA Taq™ is demonstrated in this figure, which shows good amplification efficiency of a 21.5 kb human genomic DNA fragment Amplification efficiency of a 21.5 kb with various amounts of template. Lane 1 = 500 ng; Lane 2 = 50 ng; Lane genomic DNA fragment using Takara 3 = 5 ng. Lane M = λ Hind III MW marker. LA Taq™ was compared utilizing vari- ous amounts of human genomic DNA template. 6 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com Amplification of GC-Rich Templates LA Taq™ with GC buffers: Amplification of templates with high GC content or secondary structure Takara’s LA Taq™ DNA Polymerase with GC Buffers includes two dif- ferent specialized buffers (GC Buffer I and II) which can be used to facilitate amplification of templates with high GC content and/or sec- ondary structure. These buffers were developed and optimized for use with LA Taq™ to address polymerase slippage on GC-rich tem- plates and amplification problems associated with hairpin loops and other DNA secondary structures. Users are encouraged to test both buffers to see which formulation provides the best results with their template. GC Buffer I and II are also supplied as part of the compre- hensive LA PCR Amplification Kit, which additionally contains Mg2+- plus and Mg2+-minus LA Buffer II, 25 mM MgCl2, Control DNA, and MW markers. ■ This figure demonstrates the performance of the LA Taq™ DNA Amplification of a portion of Polymerase with GC Buffers in amplification of a portion of the Huntington's Disease (IT 15 CAG Huntington’s Disease gene (IT 15 CAG repeat). Two DNA fragments repeat) using LA Taq™ with GC were amplified using LA Taq™ with either LA Buffer II (lane 1), GC Buffers. Buffer I (lane 2), or GC Buffer II (lane 3). The GC content of the 262 bp fragment is 73%; the GC content of the 358 bp product is 71.5%. a GC Kit from company A is in lane 4. Lane M contains a 100 bp molecular weight ladder. Sensitive, Specific Long PCR LA Taq™ Hot Start: Facilitates room temperature assembly 1 2 3 LA Taq™ Hot-Start DNA Polymerase consists of LA Taq™ DNA Polymerase plus a monoclonal Taq antibody bound to the poly- merase. It retains all of the high performance features of LA Taq™, and because the enzyme is sequestered by the antibody until the first denaturation step, it additionally provides increased reaction specifici- ty and reduced background. ■ This figure shows amplification of a long (15 kb) λ DNA fragment using either LA Taq™ (lane 2) or LA Taq™ Hot-Start (lane 3) DNA Polymerase. Use of LA Taq™ Hot-Start prevented the non-specific product amplification observed with LA Taq™. In addition, room temperature reaction assembly, (which is especially important in automation), is possible with this formulation. Amplification of a 15 kb λ DNA fragment using LA Taq™ DNA Polymerase (Lane 2) and LA Taq™ DNA Polymerase, Hot Start Version (Lane 3). Lane 1 contains a λ Hind III MW marker. 7 Takara Mirus Bio • 505 S. Rosa Road, Suite 101, Madison WI, 53719 • 888-251-6618 • www.takaramirusbio.com Application Recommended Product Efficient and Sensitive PCR Ex Taq™ DNA Polymerase, Ex Taq™ Hot Start DNA Polymerase Long and Accurate PCR LA Taq™ DNA Polymerase, LA Taq™ Hot Start DNA Polymerase High Fidelity PCR LA Taq™ DNA Polymerase, Ex Taq™ DNA Polymerase Hot-Start PCR Ex Taq™ Hot Start DNA Polymerase, LA Taq™ Hot Start DNA Polymerase Real-Time PCR SYBR® Premix Ex Taq™ with ROX, Ex Taq™ R-PCR DNA Polymerase, Premix Ex Taq™ (Perfect Real Time) Automated PCR Ex Taq™ Premix, Taq Premix, One Shot LA Taq ™ Mix, Premix Ex Taq™ (Perfect Real Time) Difficult templates Ex Taq™ DNA Polymerase, Ex Taq™ Hot Start DNA Polymerase GC-rich or High Secondary Structure LA Taq™ DNA Polymerase with GC Buffers, LA PCR Kit General PCR Taq Polymerase, Ex Taq™ DNA Polymerase Ordering Information Ex Taq™ Products Product No. Description Quantity Price TAK RR001A Ex Taq™ DNA Polymerase 250 units (200 reactions) $ 158.00 TAK RR001B Ex Taq™ DNA Polymerase 1,000 units (800 reactions) $ 567.00 TAK RR001C Ex Taq™ DNA Polymerase 3,000 units (2400 reactions) $ 1483.00 TAK RR01AM Ex Taq™ DNA Polymerase (Mg2+-free Buffer) 250 units (200 reactions) $ 158.00 TAK RR01BM Ex Taq™ DNA Polymerase (Mg2+-free Buffer) 1,000 units (800 reactions) $ 567.00 TAK RR01CM Ex Taq™ DNA Polymerase (Mg2+-free Buffer) 3,000 units (2400 reactions) $ 1483.00 TAK RR003 Ex Taq™ DNA Polymerase Premix 500 µl x 6 (120 reactions) $ 147.00 TAK RR006A Ex Taq™ Hot Start DNA Polymerase 250 units (200 reactions) $ 189.00 TAK RR006B Ex Taq™ Hot Start DNA DNA Polymerase 1,000 units (800 reactions) $ 666.00 TAK RR031A Ex Taq™ R-PCR Enzyme-Buffer system, Version 2.1 250 units (200 reactions) $ 189.00 TAK RR031B Ex Taq™ R-PCR Enzyme-Buffer system, Version 2.1 1,000 units(800 reactions) $ 666.00 TAK RR039A Premix Ex Taq™ (Perfect Real Time) DNA polymerase 200 reactions $ 219.00 TAK RR039B Premix Ex Taq™ (Perfect Real Time) DNA polymerase 400 reactions $ 437.00 TAK RR041A SYBR® Premix Ex Taq™ (Perfect Real Time) with ROX 200 reactions $ 273.00 TAK RR041B SYBR® Premix Ex Taq™ (Perfect Real Time) with ROX 400 reactions $ 534.00 LA Taq™ Products Product No. Description Quantity Price TAK RR002T LA Taq™ DNA Polymerase (Trial Size) 50 reactions $ 65.00 TAK RR002M LA Taq™ DNA™ Polymerase 250 units (100 reactions) $ 263.00 TAK RR002B LA Taq™ DNA Polymerase 1000 units (400 reactions) $ 979.00 TAK RR002C LA Taq™ DNA Polymerase 3,000 units (1200 reactions) $ 2781.00 TAK RR002A LA Taq™ Supplement (with Mg2+-free Buffer) 125 units (50 reactions) $ 131.00 TAK RR02AG LA Taq™ DNA Polymerase (with GC Buffers) 125 units (50 reactions) $ 131.00 TAK RR004 One Shot LA PCR Mix (with GC Buffers) 24 reactions $ 113.00 TAK RR013A LA PCR Amplification Kit, Version 2.1 50 reactions $ 221.00 TAK RR013B LA PCR Amplification Kit, Version 2.1 100 reactions $ 420.00 TAK RR042A LA Taq™ Hot Start DNA Polymerase 125 units (50 reactions) $ 171.00 TAK RR042B LA Taq™ Hot Start DNA Polymerase 500 units (200 reactions) $ 631.00 TAK RR042T LA Taq™ Hot Start DNA Polymerase (Trial Size) 50 units $ 81.00 Taq Products Product No. Description Quantity Price TAK R001A Taq DNA Polymerase 250 units $ 113.00 TAK R001B Taq DNA Polymerase 1,000 units (4 x 250 units) $ 391.00 TAK R001C Taq DNA Polymerase 3,000 units (12 x 250 units) $ 1,030.00 TAK R001AM Taq DNA Polymerase (with Mg2+-free Buffer) 250 units $ 113.00 TAK R001BM Taq DNA Polymerase (with Mg2+-free Buffer) 1,000 units (4 x 250 units) $ 391.00 TAK R001CM Taq DNA Polymerase (with Mg2+-free Buffer) 3,000 units (12 x 250 units) $ 1,030.00 TAK R004A Taq DNA Polymerase Premix 120 reactions (6 x 500 µL) $ 123.00 Ex Taq™ and LA Taq™ are trademarks of Takara Bio Inc. SYBR® is a registered trademark of Molecular Probes, Inc. Lightcycler® is a registered trademark of a member of the Roche group. RotorGene™ is a trademark of Corbett Research. Smart Cycler® is a registered trademark of Cepheid. AmpliTaq® Gold is a trademark of PE Applied Biosystems. Advantage™ is a trademark of BD Biosciences Clontech. Platinum® Taq is a trademark of Invitrogen Life Technologies. Proof-Start™ is a trademark of Qiagen, Inc. LA PCR technology is covered by U.S. Patent No. 5,436,149 issued to Takara Bio Inc. Takara Bio’s Hot-Start PCR-related products are licensed under U.S. Patent 5,338,671 and 5,587,287 and corresponding patents in other countries. Takara PCR Related Products are sold under a licensing arrangement with Roche Molecular Systems and F. Hoffman La Roche Ltd. and Applied Biosystems. Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process (and RT or other as appropriate) in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as pur- chased, ie., an authorized thermal cycler.
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