Omni Klentaq Enables Direct DNA Amplification

Transcription

1 Omni Klentaq Enables Direct DNA Amplification Inhibition Resistant, High Performance, Hot-Start Taq DNA Polymerase TOLERANT of up to 40% whole blood; also tolerates inhibitors in soil, foods, and intercalating dyes. ENABLES DIRECT PCR: No DNA extraction needed; increases throughput, lowers cost, minimizes sample loss. HOT-START: Engineered to dramatically reduce activity during room temperature reaction set-up. PROVEN: Applications include clinical diagnostics, forensics, environmental and food testing. COST EFFECTIVE: Low-cost, simple solution that reduces overall PCR costs.

2 Omni Klentaq DNA Polymerase Product Overview The premier enzyme for clinical, forensic and surveillance PCR applications Figure 1: Location of the Mutagenized Codons in Klentaq. Crystal structure of engineered Omni Klentaq molecule emphasizes the location of the mutated residues. Upper right is the thumb domain, upper left is the fingers domain, where the mutations can be seen at the surface of the hinge point (knuckles) of the fingers. Primer and template DNA strands are rendered in stick mode (green) in the active site cleft. Since the discovery of PCR, DNA amplification has been a powerful identification tool in diagnostic and surveillance settings, while sample preparation has been an obstacle to widespread commercial success. Omni Klentaq, a novel, Taq DNA polymerase-derived enzyme, has removed the need for costly sample pre-treatment by significantly improving tolerance to the inhibitors present in bodily fluids, soil, water, and foods, making it the ideal choice for PCR identification applications around the globe. Omni Klentaq is a truncated (N-terminal deletion), Taq DNA polymerase variant lacking proofreading (3-5 ) and nick translation (5-3 ) nuclease functions which has been engineered to enhance its resistance to common environmental inhibitors. An additional series of mutations provides a fast activating, on-board hot-start function. Game-Changing Performance Through Simplicity Omni Klentaq is recommended for use in PCR assays where sample complexity is a challenge and time-toresult is critical. This superior enzyme is designed to perform when standard PCR enzymes fail, and simplify clinical diagnostic and surveillance PCR, resulting in an enzyme which combines novel features including: Page 2 Tolerance of common PCR inhibitors Elimination of DNA extraction steps Improved specificity & sensitivity Low-cost Hot Start without additives Up to 5X higher tolerance of DNA intercalating dyes Omni Klentaq

3 Ready for the Clinic: Quality, Consistency, Scalability In addition to these key performance features, Omni Klentaq benefits from Enzymatics world-renowned record of success producing benchmark-quality enzyme products under its ISO 13485:2003 Quality System. This ensures that the product is ready to meet the rigorous demands of global supply, and well-matched to any regulatory submission. Cold-Sensitive Mutant Taq Maximizes Hot-Start Convenience Commercial PCR presents several challenges that are best addressed by a polymerase which is inactive at ambient temperatures. Omni Klentaq incorporates a simple, intrinsic, hot-start feature via mutation of the enzyme that suppresses its activity at low temperatures. This reduces Omni Klentaq s ability to synthesize DNA at temperatures encountered during formulation, dry-down, dispensing, and reaction setup, resulting in dramatically lower mispriming and primerdimer formation. This novel hot-start approach is: FAST Activation is instant REVERSIBLE Activity is modulated by temperature SIMPLE No hot-start additives necessary LOW COST No additional manufacturing or license costs Omni Klentaq provides the lowest possible cost hot-start performance without any additives or changes to the PCR protocol. Omni Klentaq Fast Start Taq Platinum Taq Manual Hot Start > No No Yes No Yes Figure 2: Intrinsic Hot-Start Performance of a Double Cold-Sensitive Taq Mutant, Omni Klentaq. Amplification of two difficult, highly CG-rich targets of mouse rrna genes (lanes 1 and 2), with Omni Klentaq, Fast Start Taq, and Platinum Taq, with or without manual hot start (addition of Mg at high temperature). Page 3

4 Resistant to PCR Inhibitors in Blood Direct PCR for Diagnostics Omni Klentaq - PCR Directly from Blood Samples Whole blood contains a variety of substances known to inhibit PCR, an issue which has complicated diagnostic and forensic applications for decades. This serious problem elevates the time and cost of addressing clinically important human health issues such as the diagnosis of genetic and infectious disease, blood typing, and monitoring the safety of the public blood supply. With its ability to amplify DNA in the presence of a wide variety of inhibitory substances, Omni Klentaq is available to enable a new generation of time and cost-efficient diagnostic PCR applications. Omni Klentaq benefits clinical diagnostic PCR by: Reducing time-to-answer by minimizing sample pre-treatment steps Simplifying processing, allowing a lesser degree of operator training Increasing instrument throughput Decreasing variable (labor and consumables) and fixed (time on instrument) costs Omni KlenTaq JumpStart Taq DNA Blood DNA Blood M N N M M N N M AmpliTaq Gold DNA Blood M N N M Figure 3: Comparison of Omni Klentaq vs. Commercial Enzymes in Amplification of Hepatitis B Virus from Whole Blood Samples. A 690 bp target from Duck hepatitis B virus (DHBV) P gene was amplified from five five-fold dilutions of DNA, kitpurified from duck serum (lanes 1-5), or five equivalent amounts of duck serum mixed with whole human blood (10%), using Omni Klentaq, JumpStart Taq, or AmpliTaq Gold enzymes. Results: With Omni Klentaq, the test target was detected with similar efficiency both from purified DNA and blood. In contrast, JumpStart Taq produced very faint bands at higher concentrations of template, while AmpliTaq Gold completely failed to work with blood and practically both enzymes were dependent on DNA purification prior to PCR. Page 4 Omni Klentaq

5 Omni Klentaq s performance in PCR of blood samples versus that of other commercial enzymes is shown in Figure 3 on the previous page. Omni Klentaq is the only enzyme able to amplify the target DNA in the presence of whole blood, thereby eliminating the need for a DNA purification step. In addition to enabling highperformance diagnostic PCR from whole blood, Omni Klentaq also exhibits performance benefits across a spectrum of clinical specimens, including urine, stool, plasma, serum, and sperm samples. Real time PCR of Inhibitor-Rich Samples Perhaps the most challenging application in diagnostic PCR is the pairing of quantitative, intercalating dye-based detection chemistry with complex clinical samples. Omni Klentaq can improve the results of methods which rely on intercalating dyes for amplicon detection regardless of the sample type/origin. These dyes are known to suppress the activity of Taq-based enzymes, and this effect is compounded by the presence of inhibitor-rich samples such as whole blood, which may require an increased dye concentration to overcome attenuation of the fluorescence signal caused by the sample. While most commercial Taq-derived PCR polymerases can tolerate less than 0.5-1x the recommended SYBR Green concentration in PCR, Omni Klentaq remains functional even in the presence of 4-5X the standard concentration of SYBR Green I dye. Tolerant of Inhibitors in Water Samples Detect and Quantitate Pathogens Directly Due to the sensitivity and specificity of PCR, there is growing interest in using PCR-based methods to detect and quantitate pathogens directly, especially pathogens that are difficult to culture. However, a major impediment to more widespread use of PCRbased methods is the presence of inhibitors that frequently contaminate DNA isolated from environmental sources, especially water rich in organic matter. Omni Klentaq is resistant to such inhibitors and facilitates direct PCR in water samples, saving both time and money. Page 5

6 Soil Inhibition Resistant Low Cost Sensitive Detection of Soil-born Microorganisms Omni Klentaq, with its proven ability to overcome inhibitors, can benefit forensic PCR applications, where samples are frequently collected from soil substrates. Direct PCR of crude soil extracts is highly problematic, due to the presence of humic acid, regarded as the most potent soil inhibitor to PCR analysis. As Taq polymerase is completely inhibited by trace amounts of humic acid, DNA from soil must be purified prior to PCR.As shown in Figure 5, Omni Klentaq exhibits high efficiency in the presence of humic acid, outperforming competing Taq enzymes. This capability enables low cost and highly sensitive detection of soil-borne targets. Fast Start Taq Jump Start Taq Omni KlenTaq Fast Start Taq Jump Start Taq Omni KlenTaq M M Figure 5: Omni Klentaq Outperforms Commercial Taqs in Direct PCR Detection of Soil-Born Microorganisms in Crude Soil Samples. Two commercial Taq enzymes, Fast Start Taq (Roche), Jump Start Taq (Sigma), and our Omni Klentaq mutant enzyme were used to amplify an endogenous 600 bp target of Bacillus Cereus from a crude soil extract. The reactions contained four dilutions of the soil extract: 16%, 8%, 4% and 2% (lanes 1-4). Reactions in lanes 5 were positive controls, containing 5 ng purified B. cereus DNA instead of soil extract. PCR was performed in real-time cycler Opticon-2, and the amplified products were analyzed both by SYBR Green dye fluorescence (top panels) and gel electrophoresis (bottom panels). The pink curves correspond to the control reactions and the yellow, blue, green and red ones reflect the reactions with increasing soil concentration. Page 6 Omni Klentaq

7 Robust PCR in Crude Milk Samples Fast and Accurate Results The need for low-cost, high accuracy diagnostic testing has increased as Industrial food production scales. Omni Klentaq is unique in its ability to enable rapid, inexpensive diagnostic testing at food manufacturing sites. An example of Omni Klentaq s unparalleled ability to deliver high performance, low cost results is in the dairy industry. Here, PCR is widely used to control for many factors including authenticity of dairy products and pathogens. The proteins, calcium ions and fats in milk samples interfere with PCR amplification, frequently resulting in false negatives, with potentially life-threatening consequences. Omni Klentaq is resistant to these inhibitors and preforms robustly even in crude milk samples, including whole milk, as show in Figure 6. Fat free Milk 2% Fat Milk Whole Milk Milk (μl / reaction) > M Omni KlenTaq KlenTaq AmpliTaq Vendor 1 Gold Figure 6: Amplification of Salmonella DNA in Crude Milk Samples. A 534 bp target of Salmonella inv gene (1 ng DNA) was amplified for 35 cycles with Omni Klentaq or AmpliTaq Gold in the presence of various amounts and types of milk (0-20 ul per 50 ul reaction). The reactions with mutant enzymes were supplemented with a novel PCR enhancer cocktail (PEC). This enhancer was not included in the AmpliTaq Gold reactions since it was not compatible with this enzyme. Page 7

8 Enzymatics : The New Standard in Enzyme Production Quality, Service, and Value You Can Count On Enzymatics was founded in 2006 with a mission to assume a global leadership position in the commercial life science marketplace for protein products and services. The Company partners with its customers to resolve protein-related challenges by providing the highest quality products and services available, delivered under ISO compliance, and at paradigm-shifting prices. Our focus is centered on partnering to deliver unique, creative solutions to customer challenges, enabling them to focus their precious resources on the development and commercialization of world-class technologies. Enzymatics introduction of our elite-grade Omni Klentaq DNA Polymerase is the result of careful analysis of the needs of our diagnostic-sector customers and furthers our commitment to identifying, acquiring, and developing the very best protein technologies available. If your company has a requirement for a protein products and services partner that stands clearly above the crowd, we want to hear from you. For more infomation please contact us: Enzymatics Inc. 100 Cummings Center Suite 407J Beverly, MA Phone (888) Fax (978) Limitations of Use: This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. MSDS sheets relevant to this product are available upon request Enzymatics Inc. All rights reserved. Enzymatics is a registered trademark of Enzymatics Inc. This product is sold under a license from DNA Polymerase Technology, Inc., U.S.Patent Nos. 7,462,475 and 5,436,149. Omni Klentaq is a registered trademark of DNA Polymerase Technology, Inc