PCR enzyme for your application?

Transcription

1 AMPLIFICATION PCR and RT-PCR Reagents How do you ensure optimal amplification results? How do you choose the best PCR enzyme for your application? How can you improve cdna synthesis?

2 PCR AND RT-PCR REAGENTS We have the best PCR reagent for your application. Our expertise in enzyme engineering has produced one of the broadest portfolios of high-fidelity PCR enzymes and reverse transcriptases available today. This expertise has translated to advances in high-fidelity reverse transcription PCR. Whether you need the highest accuracy available, large product yields, or sensitive cdna synthesis, trust our high-performance enzymes to answer your amplification challenges. CONTENTS: PCR Enzymes & Master Mixes 2-3 PCR and RT-PCR Reagents Selection Guide 4 Pfu-Based Fusion DNA Polymerases 5 Ultra-High Fidelity and Fast PCR 6 Superior Yield and GC-Rich s 7 Ultra-High Fidelity PCR 8 High Fidelity for TA/UA Cloning 9 High Fidelity for Blunt-End Cloning 10 High Sensitivity & Economy 11 Long PCR RT-PCR Kits & Reagents 12 High Fidelity RT-PCR Cloning 13 High Yield Multi-Temperature RT PCR Cloning System 14 PCR Cloning Kit 15 Ordering Information

3 The Importance of High Fidelity High fidelity PCR enzymes are valuable for minimizing the introduction of amplification errors in products that will be cloned, sequenced, and expressed. Significant time and effort can be saved by employing high fidelity amplification procedures that eliminate the need for downstream error-correction steps and can minimize the number of clones that must be sequenced in order to obtain error-free constructs or accurate consensus sequences. Moreover, the use of high fidelity amplification conditions is essential when analyzing small amounts of template DNA or rare molecules in heterogeneous populations. Amplifications employing small amounts of template DNA are especially prone to high mutant frequencies due to PCR-generated errors in early cycles 1, 2. The ArchaeMaxx Factor Advantage Because proofreading PCR enzymes can be finicky, we have improved high fidelity PCR to be more reliable. Our ArchaeMaxx Polymerase-Enhancing Factor, found exclusively in our high fidelity DNA polymerases, improves overall PCR performance by eliminating the inhibition of proofreading enzymes caused by incorporation of dutp, which results from deamination of dctp during PCR 3. Our PCR enzymes formulated with the ArchaeMaxx factor produce higher yields and exhibit greater target length capability. Superior Reverse Transcription Our expertise in enzyme engineering also extends to reverse transcriptases (RT). Both MMLV and AMV reverse transcriptases possess substantial RNase H activity that degrades RNA molecules and limits full-length cdna synthesis and yield. For this reason, RTs that lack RNase H activity are far superior to MMLV and other RNase H RTs. Our StrataScript 5.0 Multiple Temperature Reverse Transcriptase and AccuScript High Fidelity Reverse Transcriptase allow more efficient and accurate production of full-length cdna.

4 2>3 PCR AND RT-PCR REAGENTS SELECTION GUIDE PCR Enzymes PfuUltra II Fusion HS DNA Polymerase Application High Fidelity Cloning and Mutagenesis Accuracy Length vs. Taq 20x Reliability PfuUltra High Fidelity DNA Polymerase High Fidelity Cloning and Mutagenesis 18x PfuTurbo DNA Polymerase High Fidelity Cloning and Mutagenesis 6x Pfu DNA Polymerase, cloned or native High Fidelity Cloning and Mutagenesis 6x Herculase II Fusion DNA Polymerase Superior Yield / GC-Rich s 6x Herculase Enhanced DNA Polymerase Amplifying Difficult / GC-Rich s 3x Easy-A High Fidelity PCR Cloning Enzyme High Fidelity TA / UA Cloning 6x EXL DNA Polymerase Amplifying Long s 3x PicoMaxx High Fidelity PCR System Sensitivity / Economy 2x RT-PCR KITS & REAGENTS COMPARISON OF O Format High Yield/Sensitivity High Fidelity Cloning RT-PCR Reagent cdna Synthesis One-Tube RT-PCR StrataScript 5.0 Multiple Temperature RT StrataScript One-Tube RT-PCR Kit with Easy-A PCR Cloning Enzyme AccuScript High Fidelity First Strand cdna Synthesis Kit StrataScript One-Tube RT-PCR Kit with Easy-A PCR Cloning Enzyme AccuScript High Fidelity AccuScript High Fidelity AccuScript High Fidelity Two-Tube RT-PCR StrataScript 5.0 Multiple Temperature RT plus PicoMaxx PCR System AccuScript High Fidelity RT-PCR cdna Synthesis Kit (includes PfuUltra DNA Polymerase StrataScript 5.0 Multiple StrataScript First-Strand StrataScript First-Strand plus PicoMaxx High Fide StrataScript One-Tube RT with Easy-A PCR Cloning

5 Yield Speed Length 0-19 kb (genomic) 0-20 kb (vector) kb (cdna) 0-17 kb (genomic) 0-20 kb(vector) kb (cdna) 0-19 kb (genomic) 0-20 kb (vector) kb (cdna) Blunt or 3 -A ends Blunt Blunt Blunt Exclusive ArchaeMaxx Advantage HotStart Master Mix 0-10 kb Blunt 0-12 kb (genomic) 0-15 kb (vector) kb (cdna) Blunt 0-37 kb Mixed 0-5 kb 3 -A kb (genomic) kb (vector) Mixed 0-10 kb kb (cdna) Mixed R RT-PCR KITS AND REAGENTS Length RNase H Deficient Non-Specific Activity Endonuclease Activity cdna Synthesis Attribute One-Tube RT-PCR Format Two-Tube RT-PCR Format Reverse Transcriptase 0-20 kb Fidelity First-Strand cdna Synthesis Kit 0-20 kb Fidelity RT-PCR Kit 0-9 kb Fidelity Temperature Reverse Transcriptase 0-20 kb Yield at Multiple Temperatures Synthesis Kit 0-9 kb Yield Synthesis Kit lity PCR System 0-9 kb Yield -PCR Kit Enzyme 0-5 kb Yield

6 4>5 PCR AND RT-PCR REAGENTS > PCR ENZYMES & MASTER MIXES Pfu-Based Fusion DNA Polymerases A New Generation of Fast, High Fidelity DNA Polymerases Our new generation of high fidelity fusion DNA polymerases feature improved processivity and, together with exclusive enzyme improvement additives, provide superior yield, better reliability, and super-fast cycling times. With the demanding needs for your cloning, sitedirected mutagenesis, and expression applications, our high-fidelity fusion DNA polymerases provide you with unsurpassed performance. A New Standard in High Fidelity PCR Performance Our high fidelity fusion DNA polymerases set a new standard in high-fidelity PCR performance. We have upgraded the processivity of our Pfu-based enzymes by fusing our PfuUltra and Herculase DNA polymerases with a high affinity double-stranded DNA binding domain. This domain serves to better anchor the DNA polymerase, preventing early dissociation from the DNA template. The enhanced processivity and exclusive PCR performance additives of our high fidelity fusion DNA polymerases provide you with superior yield, excellent reliability and much shorter overall run time. Enhanced Processivity and Improved Template Integrity The processivity of our PfuUltra II fusion HS DNA polymerase has improved 12-fold over the Pfu DNA polymerase (Table 1). The PfuUltra II enzyme is also shown to be over 5-fold more processive than other fusion enzymes including Phusion, iproof, and PfxUltima. This improved processivity of the PfuUltra II and Herculase II enzymes allows them to incorporate more nucleotides per binding event, enhancing PCR yields and shortening extension times, hence saving you time and improving template integrity by minimizing exposure to extreme cycling temperatures. We offer two new high fidelity fusion DNA polymerases with different key features: The PfuUltra II Fusion HS DNA Polymerase a,b for ultra-high fidelity The Herculase II Fusion DNA Polymerase a,b for superior yields and GC-rich targets. Extreme Speed The enhanced processivity of our PfuUltra II and Herculase II high fidelity fusion DNA polymerases promotes much shorter extension times and more robust, high yield amplifications. While the typical proofreading polymerases require 1-2 minutes per kilobase extension times depending upon the target length, PfuUltra II and Herculase II fusion DNA polymerases perform exceptionally well with short extension times in "seconds per kilobase. For example, the Herculase II fusion DNA polymerase produced superior yield of a 900 bp human α1at gene fragment with 1 second extension time (Figure 1). Enzyme Median Processivity (nt) Processivity vs. Pfu (fold) 1 second extension 3 second extension 5 second extension 10 second extension PfuUltra II Fusion HS DNA Polymerase kb Phusion DNA Polymerase PfxUltima DNA Polymerase Cloned Pfu DNA Polymerase Table 1 ENHANCED PROCESSIVITY OF THE PfuUltra II FUSION HS DNA POLYMERASE Processivity of each enzyme was assayed with manufacturer s recommended buffer. Figure 1 DRAMATICALLY REDUCED EXTENSION TIME The Herculase II Fusion DNA Polymerase amplified a 900 bp human α1at gene fragment from genomic DNA with as short as 1 second extension time.

7 Ultra-High Fidelity and Fast PCR PfuUltra II Fusion HS DNA Polymerase We are the leading developer of high fidelity PCR enzymes. In our PfuUltra II Fusion HS DNA Polymerase, we coupled polymerase fusion technology with our engineered PfuUltra high fidelity DNA polymerase, hotstart antibody, and proprietary ArchaeMaxx PCR enhancing factor. This provides you with extreme accuracy, high specificity, and long target-length capability, while dramatically reducing the overall PCR extension times. Industry-Leading Fidelity Our PfuUltra II Fusion HS DNA Polymerase is the new industry standard for ultra-high fidelity PCR. Our data demonstrates that the PfuUltra II fusion HS DNA polymerase exhibits an accuracy of 3-fold higher than any other high fidelity enzyme and 20-fold higher than Taq DNA Polymerase, making it the most accurate enzyme available (Figure 2). PCR Enzyme PfuUltra II Fusion HS DNA Polymerase Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage Hot Start 0-19 kb (genomic) 20x 0-20 kb (vector) Blunt kb (cdna) Master Mix Amplify Long s in a Fraction of the Time The PfuUltra II fusion HS DNA polymerase produces higher yields under fast cycling conditions. It amplifies genomic targets up to 19 kb in length (Figure 3). In contrast, most other commercial fusions exhibit limited target-length capability, such as up to 6 kb. It is critical to use high fidelity enzymes for amplifying long targets, since mutation frequency increases linearly with amplicon size. High Specificity Hotstart Version Unlike other commercial fusion DNA polymerases, our PfuUltra II DNA polymerase is formulated with hotstart (HS) antibodies that neutralizes both polymerase and exonuclease activities during reaction setup, thereby enhancing specificity and facilitating robotic assemblies Accuracy of Proofreading DNA Polymerase PfuUltra II Fusion HS DNA Polymerase Enzyme Accuracy (x10 5 ) Kb PfuUltra II Enzyme Herculase II Enzyme Phusion / DeepVent Vent Pfx/KOD Pfx50 Taq iproof Figure 2 PfuUltra II FUSION HS DNA POLYMERASE, THE HIGHEST FIDELITY PCR ENZYME The PfuUltra II Fusion HS DNA Polymerase exhibits accuracy 3-fold higher than the Phusion and iproof DNA polymerases and 20-fold higher than Taq DNA Polymerase. Fidelity was measured using our validated and referenced fidelity assay 4. (Accuracy = 1/error rate.) Figure 3 THE PfuUltra II FUSION HS DNA POLYMERASE AMPLIFIES A WIDE RANGE OF TARGETS The PfuUltra II Fusion HS DNA Polymerase amplifies a broad range of genomic DNA targets up to 19 kb in length.

8 6>7 PCR AND RT-PCR REAGENTS > PCR ENZYMES & MASTER MIXES Superior Yield and GC-Rich s Herculase II Fusion DNA Polymerase The Herculase II Fusion DNA Polymerase, with its special formulation and the addition of our exclusive ArchaeMaxx factor, provides you with superior yield for your routine PCR amplification and fast cycling times. Our Herculase II Fusion DNA Polymerase also helps you overcome challenging PCR with successful amplification of complex and GC-rich templates. In addition, our Herculase II Fusion DNA Polymerase provides accuracy comparable to Pfu DNA polymerase. PCR Enzyme Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage Hot Start Format Master Mix Herculase II Fusion DNA Polymerase 0-12 kb (genomic) 6x 0-15 kb (vector) Blunt kb (cdna) Superior Yield for Routine Applications The Herculase II fusion DNA polymerase is more robust than other PCR enzymes because of its special enzyme formulation and our exclusive ArchaeMaxx factor. In amplifications of midlength (1.7 kb) or long (6 kb) genomic fragments, our Herculase II fusion DNA polymerase produced superior yields with extension times as short as 15 sec/kb (Figure 4). Other fast or highyield proofreading DNA polymerases failed to amplify or produced lower yield. Difficult/GC-Rich s The Herculase II fusion DNA polymerase is also ideal for the amplification of difficult-to-amplify targets. The enhanced processivity of the enzyme, the exclusive enzyme improvement additives, and optimized buffer system enable the enzyme to amplify difficult templates such as GC-rich targets. It easily amplifies targets with as high as 84% GC content (Figure 5). DMSO is provided separately as a PCR adjunct, and can be added when amplifying difficult targets. Herculase II Enzyme Phusion /iproof Pfx50 Pwo SuperYield Seconds / kb Herculase II Enzyme IGFB FMR1 HTR MMZ5 kb Figure 4 ROBUST YIELDS ACHIEVED WITH FAST CYCLING TIMES The Herculase II Fusion DNA Polymerase produced superior yields of a 6 kb fragment in amplification employing human genomic DAN and extension times of 15, 30, 45, and 60 sec/kb. Experiments were conducted under identical conditions using each enzyme s recommended buffer. Figure 5 THE HERCULASE II FUSION DNA POLYMERASE EXCELS IN AMPLIFYING GC-RICH TARGETS The Herculase II Fusion DNA Polymerase easily amplifies GC-rich fragments from human genomic DNA. FMR1, Fragile X mental retardation syndrome protein (84% GC); MML, D - amino acid oxidase activator (35% GC); MMZ5, ZIC5 - zinc family member 5 protein (68% GC).

9 Ultra-High Fidelity PCR Accurate PCR Enzyme in Convenient Master Mix Format Our continuing efforts to improve the fidelity of PCR have resulted in the PfuUltra High Fidelity DNA Polymerase a,b. The PfuUltra DNA polymerase is ideal for PCR cloning, site-directed mutagenesis, and anywhere sequence accuracy is critical for downstream applications. Ultra High Fidelity The PfuUltra High Fidelity DNA Polymerase contains a genetically engineered mutant of Pfu DNA polymerase a,b,c that delivers 300% greater accuracy. A validated and referenced fidelity assay 4 demonstrates that the PfuUltra high fidelity DNA polymerase exhibits an average error rate 3-fold lower than Pfu DNA polymerase and 18-fold lower than Taq DNA polymerase (Figure 2). Enhanced Performance In addition to ultra-high fidelity, our PfuUltra high fidelity DNA polymerase exhibits superior PCR performance compared to most PCR proofreading enzymes. The addition of our ArchaeMaxx polymerase-enhancing factor promotes higher yields, shorter extension times, and greater target length capability (Figure 6). PCR Enzyme PfuUltra High Fidelity DNA Polymerase Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage Hot Start High Specificity Hotstart Version The PfuUltra Hotstart DNA Polymerase a,b is an antibody-mediated hotstart formulation of the PfuUltra high fidelity DNA polymerase that provides higher specificity and reduced background. Convenient Master Mix Formulation For ultra-high fidelity with added convenience and increased throughput, choose our PfuUltra Hotstart PCR Master Mix a,b. This 2x formulation contains the PfuUltra hotstart DNA polymerase, buffer, magnesium, and dntps all in one tube for faster setup and more consistent results. Master Mix 0-17 kb (genomic) 18x Blunt 0-15 kb (vector) M kb Figure 6 AMPLIFIES A WIDE RANGE OF TARGETS The PfuUltra High Fidelity DNA Polymerase amplifies genomic DNA targets up to 17 kb.

10 8>9 PCR AND RT-PCR REAGENTS > PCR ENZYMES & MASTER MIXES High Fidelity for TA/UA Cloning Unique Proofreading Formulation Adds A-Overhangs TA or UA cloning requires PCR products with 3'-A overhangs. We created the Easy-A High Fidelity PCR Cloning Enzyme a,d,e to automatically add A-overhangs for quick and easy high fidelity cloning into TA/UA vectors. PCR Enzyme Easy-A High Fidelity PCR Cloning Enzyme Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage Hot Start Format Master Mix 6x 0-5 kb 3'-A Combines Cloning Efficiency with High Accuracy The Easy-A High Fidelity PCR Cloning Enzyme is the only proofreading DNA polymerase formulation to deliver highthroughput TA or UA cloning. PCR products amplified with the Easy-A PCR cloning enzyme can be cloned directly to the StrataClone PCR Cloning Vector with high cloning efficiency and throughput (Figure 7). But unlike Taq DNA polymerase, the Easy-A PCR cloning enzyme exhibits proofreading activity that provides the accuracy level of Pfu DNA polymerase. Fidelity Without Extra Work Adapting blunt-ended fragments amplified with proofreading enzymes requires post-pcr addition of 3'-A overhangs prior to the cloning step. This additional step includes a secondary incubation with Taq DNA polymerase and requires the opening and closing of tubes, which exposes the lab to contamination risk. The Easy-A high fidelity PCR cloning enzyme does not require any post-pcr A-addition steps. High Specificity Hotstart Formulation The Easy-A high fidelity PCR cloning enzyme is provided as an antibody-mediated hotstart formulation, providing higher specificity and reduced background. Convenient Master Mix Formulation For the highest throughput in TA or UA cloning, choose our Easy-A High Fidelity PCR Master Mix a,d,e. This 2x formulation contains the Easy-A PCR cloning enzyme, buffer, magnesium, and dntps all in one tube for faster setup and more consistent results. Incubate PCR product with Topoisomerase I-charged vector arms (5 minutes) A PCR Product A Topoisomerase I loxp puc ori P lac lac Z' MCS U* U* MCS lac Z' ampicillin loxp Topoisomerase I Figure 7 HIGH CLONING EFFICIENCY WITH THE EASY-A PCR CLONING ENZYME AND THE STRATACLONE PCR CLONING KIT s amplified with the Easy-A High Fidelity PCR Cloning Enzyme contain 3'-A overhangs, allowing quick and easy cloning into the StrataClone PCR cloning vector with DNA topoisomerase I technology.

11 High Fidelity for Blunt-End Cloning Accuracy with Enhanced Performance We invented the high fidelity PCR market over a decade ago with the original Pfu DNA Polymerase. Since that time, our expertise has led to improvements in high-fidelity performance with the PfuTurbo DNA Polymerase a,b and, more recently, the PfuTurbo C x Hotstart DNA Polymerase a,f. These improvements have made high-fidelity PCR more reliable and versatile. Robust High-Fidelity Amplification The PfuTurbo DNA polymerase is a special formulation of cloned Pfu DNA polymerase and our ArchaeMaxx polymeraseenhancing factor that produces increased PCR product yields without affecting fidelity. The enhanced performance of our PfuTurbo DNA polymerase allows the use of shorter extension times, longer targets, and lower concentrations of DNA template than are suitable for Pfu DNA polymerase. High Specificity Hotstart Version The PfuTurbo Hotstart DNA Polymerase a,b is an antibodymediated hotstart formulation of our PfuTurbo DNA polymerase that provides higher specificity, reduced background, and enhanced yield of challenging systems. New Mutant Overcomes Uracil Sensitivity Heat can cause deamination of cytosine to uracil in a DNA strand. To prevent replication of such a mutation, archaeal (proofreading) polymerases typically stop replication just before reaching a uracil residue in the template 5. In PCR, this poisoning phenomenon can result in decreased or no product yield. M PfuTurbo C x AccuPrime PFX PLATINUM PFX 9 kb 6 kb 2.6 kb PCR Enzyme PfuTurbo DNA Polymerase PfuTurbo C x DNA Polymerase Pfu DNA Polymerase, cloned or native Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage We formulated the PfuTurbo C x hotstart DNA polymerase with a novel mutant of Pfu DNA polymerase that can read through a uracil located in the template strand without stalling. Thus, the PfuTurbo C x hotstart DNA polymerase improves the overall reliability of high fidelity PCR and exhibits less finicky, more robust performance (Figure 8). Hot Start Prevent Carryover Contamination Because our PfuTurbo C x hotstart DNA polymerase can replicate uracil-containing DNA, it can be used to prevent carryover contamination. This method involves the use of dutp in place of dttp in the nucleotide pool. Subsequent PCR reactions are pre-treated with Uracil-N-glycosylase (UNG) to degrade any du-containing DNA left over from a previous reaction; the UNG is then inactivated during the next denaturation step. The PfuTurbo C x hotstart DNA polymerase incorporates dutp in targets up to 6 kb in length more efficiently than Taq DNA polymerase. Master Mix 0-19 kb (genomic) 6x Blunt 0-15 kb (vector) 6x 0-10 kb Blunt 6x 0-5 kb Blunt 0.9 kb Figure 8 SUPERIOR PERFORMANCE WITH THE PfuTurbo C X HOTSTART DNA POLYMERASE Amplification of a range of targets from 0.9 to 9 kb using the PfuTurbo C x Hotstart DNA Polymerase and other commercially available proofreading PCR enzymes.

12 10>11 PCR AND RT-PCR REAGENTS > PCR ENZYMES & MASTER MIXES High Sensitivity & Economy Greater Performance at a Fraction of the Cost Blending Taq DNA polymerase with a proofreading enzyme improves target-length capability, fidelity, and yield, but many PCR enzyme blends lack sensitivity and can lead to PCR failures. We designed the PicoMaxx High Fidelity PCR System a,g to deliver the highest sensitivity and efficiency, bringing you greater blend performance at a much lower cost per unit than other enzyme blends. PCR Enzyme PicoMaxx High Fidelity PCR System Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage Hot Start Format Master Mix 2x 0-10 kb Mixed High Efficiency and Yield The PicoMaxx high fidelity PCR system exhibits superior amplification efficiencies due in large part to our patented ArchaeMaxx factor, which eliminates dutp that accumulates during the high heat of PCR and can poison high-fidelity reactions. Sensitivity and Reliability The PicoMaxx High Fidelity PCR System is designed to provide superior PCR sensitivity and reliability. Sensitivity is critical for successful amplification, especially with limited or precious samples; use of a PCR enzyme that lacks sensitivity often results in amplification failures. Formulated with a blend of Taq and Pfu DNA polymerases and our ArchaeMaxx polymerase-enhancing factor, together with a specially optimized buffer, the PicoMaxx high fidelity PCR system overcomes such PCR failures with greater sensitivity than other PCR enzymes (Figure 9). Superior sensitivity makes the PicoMaxx high fidelity PCR system very reliable. The ArchaeMaxx factor maximizes the performance of the proofreading component of the blend, allowing the PicoMaxx high fidelity PCR system to deliver higher product yields and more reliable performance across a wide range of targets up to 10 kb. Convenient Master Mix Formulation For superior sensitivity and yield with added convenience, choose our PicoMaxx High Fidelity PCR Master Mix a,g. This 2x formulation contains the PicoMaxx high fidelity enzyme blend, buffer, magnesium, and dntps all in one tube for faster setup and more consistent results. Genomic DNA Template Qty. (ng) PICOMAXX HIGH FIDELITY PCR SYSTEM PLATINUM TAQ HIGH FIDELITY EXPAND HIGH FIDELITY kb Figure 9 THE PICOMAXX HIGH FIDELITY PCR SYSTEM EXHIBITS SUPERIOR SENSITIVITY We amplified a 3.9 kb α-1 anti-trypsin template with the PicoMaxx High Fidelity PCR System, Expand High Fidelity PCR System (Roche), and Platinum Taq DNA Polymerase High Fidelity (Invitrogen). We performed all reactions according to the manufacturer s recommendations.

13 Long PCR Go the Full Distance To amplify very long targets, a blend of Taq DNA polymerase and a proofreading enzyme provides the best results. But not every enzyme blend can go the distance. Our EXL DNA Polymerase a,g is specially optimized to successfully and accurately amplify long targets up to 50 kb. Tackle Long Amplicons The EXL DNA Polymerase provides superior performance in amplifying complex targets greater than 20 kb in length. The EXL DNA polymerase is a special formulation of Pfu DNA polymerase, Taq DNA polymerase, and our ArchaeMaxx polymerase enhancing factor, along with a special buffer optimized specifically for very long targets. This enzyme blend provides robust yields and reliable amplification (Figure 10). PCR Enzyme EXL DNA Polymerase Accuracy vs. Taq Length Blunt or 3'-A Ends ArchaeMaxx Advantage kb (genomic) 3x Mixed kb (vector) Hot Start Master Mix Amplify with Higher Accuracy Because PCR enzymes have an intrinsic rate at which they incorporate incorrect bases, long templates are particularly prone to errors when amplified during PCR. Most long & accurate PCR enzyme blends available commercially provide only a slight increase in accuracy over Taq. Our EXL DNA polymerase provides twice the accuracy of these other blends to ensure minimal errors in your long PCR products. EXL Enzyme M COMPETITOR Figure 10 THE EXL DNA POLYMERASE EXCELS AT AMPLIFYING EXTREMELY LONG TARGETS The EXL DNA Polymerase easily amplifies two extremely long genomic targets (23 and 30 kb) and a lambda target (45 kb). A competitor s PCR enzyme for long targets is shown for comparison. All reactions were performed according to the manufacturer s recommendations.

14 12>13 PCR AND RT-PCR REAGENTS > PCR ENZYMES & MASTER MIXES High Fidelity RT-PCR Cloning Highest Accuracy RT Produces Premium cdna Our AccuScript High Fidelity Reverse Transcriptase h delivers the highest reverse transcription accuracy. This new MMLV-based RT creates cdna with 3- to 6-fold fewer errors while promoting full-length cdna synthesis and superior RT-PCR performance specifically for high fidelity cloning, protein expression or sequencing experiments. RT-PCR Reagent AccuScript High Fidelity Reverse Transcriptase AccuScript High Fidelity First-Strand cdna Synthesis Kit Length RNase H Deficient Non-Specific Endonuclease RNase Activity Activity cdna Synthesis Attribute 0-20 kb - - Fidelity 42 C 0-20 kb - - Fidelity 42 C One-Tube RT-PCR Format Two-Tube RT-PCR Format RNase H-Free Means Longer cdna Both MMLV and AMV RT possess substantial RNase H activity that degrades RNA molecules and limits full-length cdna synthesis and high yield. RT mutants lacking RNase H activity provide superior performance compared to MMLV and other RNase H RTs. To ensure maximum yields, our AccuScript RT was derived from an RNase H deficient MMLV RT mutant. AccuScript High Fidelity RT-PCR Kit - = tested to confirm absence 0-9 kb - - Fidelity 42 C Most Accurate Reverse Transcriptase Reverse transcriptases (RT) exhibit significantly higher error rates than other known DNA polymerases introducing errors at frequencies of one per 1,500 to 30,000 nucleotides during cdna synthesis 1. To solve the problem of low accuracy, we offer the AccuScript reverse transcriptase, which is an RNase H-deficient Moloney murine leukemia virus reverse transcriptase (MMLV RT) we engineered to deliver the highest transcription accuracy while promoting cdna synthesis length, yield, and RT-PCR performance. Our AccuScript RT provides over 3- to 6-fold more accurate reverse transcription than other RTs. Best Overall RT-PCR Accuracy Combining our AccuScript RT and our PfuUltra DNA polymerase lowers the incidence of errors nearly 8-fold compared to identical RT-PCR reactions performed with MMLV RT and a Taq high-fidelity blend (Figure 11). Moreover, even when cloning relatively short cdnas, the probability of recovering error-free clones is dramatically improved by replacing your standard RT-PCR reagents with our AccuScript RT and PfuUltra DNA polymerase (Figure 12). 29 Panel A Panel B Panel C No. of Clones Total No. of Mutation Sequenced Mutations Rate (per kb) 86% X Accuracy (x10 5 ) % X 1 AccuScript RT PfuUltra High Fidelity DNA Polymerase SuperScript RT Platinum Taq High Fidelity DNA Polymerase 0 AccuScript Superscript III Superscript II Transcriptor Reverse Transcriptases AMV Figure11 EIGHT-FOLD FEWER cdna ERRORS We selected and sequenced 30 random clones to determine the number of cdna clones containing errors (Panel A) and the total number of mutations (Panel B). The number of correct error-free clones (shaded bars in Panel A) using the AccuScript High Fidelity Reverse Transcriptase and our PfuUltra High Fidelity DNA Polymerase is 86% vs. only 25% using conventional error-prone RT-PCR reagents. Furthermore, the combination of the AccuScript RT and our PfuUltra DNA polymerase produced 8-fold fewer errors per kb of cdna (Panels B and C) compared to a competitor s RT-PCR method. Figure 12 THE ACCUSCRIPT REVERSE TRANSCRIPTASE FOR ERROR-FREE cdna The AccuScript Reverse Transcriptase delivers greater than 3-fold higher accuracy than other RTs. (Accuracy = 1/error rate)

15 PCR AND RT-PCR REAGENTS > RT-PCR KITS & REAGENTS High Yield Multiple Temperature RT Achieve High Yields with our Multiple Temperature Reverse Transcriptase Our new StrataScript 5.0 Multiple Temperature Reverse Transcriptase (RT) i produces high cdna yields over the broadest temperature range. It performs exceptionally well when amplifying low RNA input amounts and resolving RNA secondary structure using temperatures up to 60 C. We also offer convenient reverse transcription-pcr (RT-PCR) kits using our classic StrataScript Reverse Transcriptase i for applications such as rapid, high-fidelity TA/UA cloning. Increased Activity at All Temperatures Our StrataScript 5.0 Multiple Temperature RT is an MMLV point mutant that is engineered for improved performance over a broad range of cdna synthesis temperatures. This multitemperature capability allows you to change your RT reaction RT-PCR Reagent StrataScript 5.0 Multiple Temperature RT Length RNase H Deficient 0-20 kb - Non-Specific Endonuclease RNase Activity Activity - cdna Synthesis Attribute Yield C One-Tube RT-PCR Format Two-Tube RT-PCR Format temperature without having to change your reverse transcriptase. You are assured of high cdna yields from 40 C to 55 C, whether priming at room temperature with random hexamers, or at stringent temperatures designed to enhance priming specificity, or for transcription through GC-rich sequences. StrataScript One-Tube RT-PCR Kit with Easy-A PCR Cloning Enzyme - = tested to confirm absence 0-5 kb - - Yield 42 C Increased cdna Yield and Lower Reaction Costs The StrataScript 5.0 RT generates 2- to 4-fold more cdna than other reverse transcriptase enzymes such as SuperScript II and SuperScript III RTs (Figure 13). With low RNA input, >10-fold yield differences are achieved. Because of its high specific activity, the StrataScript 5.0 RT delivers highest cdna yields using half the amount of units per reaction than other RTs, saving you money. SuperScript III StrataScript 5.0 RT SuperScript II Two RT-PCR Systems for Cloning or High Sensitivity The StrataScript One-Tube RT-PCR System a,d combines high cloning efficiency with high fidelity in a one-tube RT-PCR format. The kit features our robust StrataScript RT and our Easy-A PCR Cloning Enzyme, which amplifies with 6-fold higher accuracy than Taq DNA polymerase, yet adds 3'-A overhangs for efficient TA or UA cloning. When sensitivity is important and you want to archive your cdna, combine our StrataScript First Strand cdna Synthesis Kit i with our PicoMaxx High Fidelity PCR System (Figure 14). Temp ( C): kb 6 kb 5 kb 4 kb 3 kb 2 kb 1.5 kb 1 kb 0.5 kb StrataScript RT PicoMaxx PCR System SuperScript II RT Platinum Taq High Fidelity DNA Polymerase Figure 13 SUPERIOR cdna YIELDS OVER A BROADER TEMPERATURE RANGE Our StrataScript 5.0 Multiple Temperature RT yielded the highest amount of cdna over a broader range of temperatures as compared to leading competitor RTs. Yield differences were most pronounced for longer (> 5 kb) cdnas, illustrating the superior length capability of our StrataScript 5.0 RT. cdna was generated from 1 µg of RNA ladder (Ambion) using equivalent units (100 units) of reverse transcriptase at various temperatures. After cdna synthesis, the samples were loaded on a 1% alkaline agarose gel. Figure 14 THE PICOMAXX PCR SYSTEM DELIVERS GREATEST SENSITIVITY IN NEW TWO-TUBE RT-PCR STRATEGY Combining the StrataScript RT with the PicoMaxx PCR System in a two tube RT-PCR format detects a viral RNA species using only 0.01 fg of input template amount.

16 14 PCR AND RT-PCR REAGENTS > PCR CLONING SYSTEM High Performance PCR Cloning StrataClone PCR Cloning Kits with DNA Topoisomerase I Technology Our new StrataClone PCR Cloning Kit h makes cloning your PCR products easier, faster, and more reliable with DNA topoisomerase I technology. Go from PCR to transformation in one short, three-step protocol guaranteed to get your clone the first time, every time. Fast, Easy, Reliable PCR Cloning Our StrataClone PCR Cloning Kits provide the highest efficiency option for topoisomerase-based PCR cloning. Cloning PCR products using DNA topoisomerase I technology saves you time and money over conventional PCR cloning with simple primer design, no PCR clean-up, and an easy three-step process. Simply add your PCR product to the vector mix, incubate 5 minutes at room temperature, and transform ligated DNA into competent cells. With our >95% clones with insert guarantee, you are sure to get your clone the first time every time. StrataClone Technology The StrataClone PCR cloning technology exploits the combined efforts of two enzymes, DNA topoisomerase I from Vaccinia virus and Cre recombinase from bacteriophage P1. In Vaccinia, DNA topoisomerase I assists in DNA replication by relaxing and rejoining DNA strands. The enzyme's site-specific endonuclease activity generates linear DNA with defined termini, and the DNA ligase activity closes the strand when replication is complete. When topoisomerase I cleaves a phosphodiester backbone of the DNA duplex, a covalent DNA - enzyme intermediate is formed, which conserves bond energy to be used for religating the cleaved DNA back to the original strand or to a heterologous DNA acceptor. The site-specific Cre recombinase enzyme catalyzes DNA recombination between asymmetric 8-bp core regions of two loxp recognition sequences. The StrataClone PCR cloning vector mix contains DNA arms charged with topoisomerase I on one end and loxp recombination sequences on the other. The topoisomerase-charged ends have a modified uridine overhang for direct ligation of Taq-amplified or Easy-A-amplified PCR products. When your PCR product is added to the mix, the DNA topoisomerase I ligates the vector arms to the PCR product resulting in a linear molecule (vector arm - PCR product - vector arm). The time required to ligate amplicon to vector is significantly reduced by the ready availability of the topoisomerase enzyme covalently bound to the vector arm. The ligated DNA is then transformed, with no clean-up steps required, into our highefficiency competent cell line engineered to transiently express Cre recombinase. The linear DNA is circularized by the Cre recombinase at the loxp sites and the recombinant vector is amplified by the host cell in an overnight incubation (Figure 15). loxp Incubate PCR product with Topoisomerase I-charged vector arms (5 minutes) puc ori P lac lac Z' Topoisomerase I A PCR Product A MCS U* U* Topoisomerase I MCS lac Z' ampicillin loxp StrataClone Competent Cells with Cre Recombinase puc ori <loxp> lac Z' MCS P lac StrataClone PCR Cloning Vector psc-a PCR Product MCS lac Z' ampicillin Figure 15 THE STRATACLONE PCR CLONING TECHNOLOGY Our StrataClone PCR Cloning System Kits exploit the combined efforts of two enzymes, Vaccinia DNA topoisomerase I and bacteriophage P1 Cre recombinase.

17 ORDERING INFORMATION Ordering Information PCR Enzymes Product Name Size Catalog Ultra-High Fidelity PfuUltra II Fusion HS DNA Polymerase 40 rxn rxn rxn PfuUltra High Fidelity DNA Polymerase 100 U U U PfuUltra Hotstart DNA Polymerase 100 U U U High Fidelity, TA or UA Cloning Easy-A High Fidelity PCR Cloning Enzyme 100 U U U High Fidelity, Blunt Cloning Pfu DNA Polymerase, Cloned 100 U U U Pfu DNA Polymerase, Native 100 U U U PfuTurbo C x Hotstart DNA Polymerase 100 U U U PfuTurbo DNA Polymerase 100 U U U PfuTurbo Hotstart DNA Polymerase 100 U U U Superior Yield and GC-Rich/Complex s Herculase II Fusion DNA Polymerase 40 rxn rxn rxn Herculase Enhanced DNA Polymerase 100 U U U Herculase Hotstart DNA Polymerase 100 U U U High Sensitivity & Economy PicoMaxx High Fidelity PCR System 100 U U U Extra-Long s EXL DNA Polymerase 100 U U U PCR Enzymes continued Product Name Size Catalog Routine PCR SureStart Taq DNA Polymerase a 100 U U U Taq2000 DNA Polymerase a 100 U U U PCR Master Mixes Ultra-High Fidelity PfuUltra Hotstart PCR Master Mix 100 rxn rxn High Fidelity, TA or UA Cloning Easy-A High Fidelity PCR Master Mix 100 rxn rxn High Fidelity, Blunt Cloning PfuTurbo Hotstart PCR Master Mix 100 rxn rxn Superior Yield and GC-Rich/Complex s Herculase Hotstart PCR Master Mix 100 rxn rxn High Sensitivity & Economy PicoMaxx High Fidelity PCR Master Mix 100 rxn RT-PCR Reagents RT-PCR Reagents Accuscript High Fidelity First Strand 50 rxn cdna Synthesis Kit Accuscript High Fidelity Reverse Transcriptase 50 rxn rxn Accuscript High Fidelity RT-PCR Kit 50 rxn StrataScript First Strand Synthesis Kit 50 rxn StrataScript One-Tube RT-PCR Kit 50 rxn with Easy-A PCR Cloning Enzyme StrataScript Reverse Transcriptase, 10,000U 50 U/µl U/µl StrataScript 5.0 Multiple Temperature 2000 U Reverse Transcriptase 10,000 U ,000 U PCR-Related Kits & Accessories PCR Cloning Kit StrataClone PCR Cloning Kit 10 rxn rxn REFERENCES 1. Hogrefe, H.H. and M.C. Borns. High Fidelity PCR Enzymes. In C.W. Dieffenbach, G.S. Dveksler (eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Cha, R.S. and W.G. Thilly. Specificity, efficiency, and fidelity of PCR. In PCR Primer: A Laboratory Manual (eds, Dieffenbach, C.W. and G.S. Dveksler) Cold Spring Harbor Laboratory Press, Hogrefe, et al. (2002). Proc. Nat. Acad. Sci.USA 99: Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: Fogg, et al. (2002). Nat Struct Biol. 9(12): Borns, M. and Hogrefe, H. (2000) Strategies. 13: 12. LEGAL Easy-A, PfuTurbo, Herculase, PicoMaxx, EXL, SureStart, and StrataScript are registered trademarks of Stratagene. PfuUltra, Taq2000, AccuScript, and StrataClone are trademarks of Stratagene. Expand is a trademark of Roche Diagnostics Corporation. Platinum is a registered trademark of Invitrogen Corporation. SuperScript is a registered trademark of Invitrogen Corporation. a. Purchase of this product is accompanied by a license under the foreign counterparts of U.S. Patents Nos. 4,683,202, 4,683,195 and 4,965,188 for use in the polymerase chain reaction (PCR) process, where such process is covered by patents, 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 purchased, i.e., an authorized thermal cycler. b. U.S. Patent Nos. 6,734,293, 6,489,150, 6,444,428, 6,379,553, 6,333,165, 6,183,997,,5,948,663, 5,866,395, 5,545,552 and patents pending c. Cloned Pfu: U.S. Patent Nos. 6,489,150, 5,948,663, 5,866,395 and 5,545,552 and patents pending Purchase of this product is accompanied by a license under the foreign counterparts of U.S. Patents Nos. 4,683,202, 4,683,195 and 4,965,188 for use in the polymerase chain reaction (PCR) process, where such process is covered by patents, 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 purchased, i.e., an authorized thermal cycler. Native Pfu: Limited Label License: Purchase of this product conveys to the purchaser only the non-transferable right under these patents to use the product for research use only by the purchaser. No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this product, either alone or as a component of another product, to any third party. Stratagene reserves all other rights, and this product may not be used in any manner other than as provided herein. For information on obtaining a license to use this product for purposes other than research, please contact Stratagene, Business Development, North Torrey Pines Road, La Jolla, California Phone (858) d. U.S. Patent Nos. 6,734,293, 6,444,428, 6,379,553, 6,333,165, 6,183,997, and patents pending e. Use of these products for certain applications may require licenses from third parties in certain countries. f. U.S. Patent Nos. 6,489,150, 5,948,663, 5,866,395, 5,545,552 and patents pending g. U.S. Patent Nos. 6,734,293, 6,489,150, 6,444,428, 6,379,553, 6,333,165, 6,183,997, 5,948,663, 5,866,395, 5,556,772, 5,545,552 and patents pending h. Patents pending i. Purchase of this PCR-related product does not convey any rights under the foreign counterparts of the PCR patents owned by Roche Molecular Systems. A license to use the PCR process, where such process is covered by patents, accompanies the purchase of certain reagents from Stratagene when used in conjunction with an Authorized Thermal Cycler.

18 AMPLIFICATION CELL BIOLOGY CLONING MICROARRAYS NUCLEIC ACID ANALYSIS PROTEIN FUNCTION & ANALYSIS QUANTITATIVE PCR SOFTWARE SOLUTIONS Stratagene USA and Canada Order: x3 Technical Services: x2 Stratagene Europe Order: Technical Services: Stratagene Japan K.K. Order: Technical Services: Distributors > For a list of worldwide distributors, please visit our website North Torrey Pines Road La Jolla, CA USA BR60-01/06