Omni Klentaq® Enables Direct DNA Amplificaon Inhibion 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.
Transcript
Omni Klentaq® Enables Direct DNA Amplification
Inhibition Resistant, High Performance, Hot-Start Taq DNA Polymerase
Figure 1: Location of the MutagenizedCodons 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.
Omni Klentaq is recommended for use in PCR assays where sample complexity is a challenge and time-to-result 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:
• 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
Game-ChangingPerformanceThroughSimplicity
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ReadyfortheClinic: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
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 set-up, resulting in dramatically lower mispriming and primer-dimer formation.
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OmniKlentaq®FastStartTaqPlatinumTaq®
ManualHotStart>NoNoYesNoYes
1 2 1 2 1 2 1 2 1 2
Figure 2: Intrinsic Hot-Start Performance of a Double Cold-Sensitive Taq Mutant, OmniKlentaq®.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).
Thisnovelhot-startapproachis:• FAST – Activation is instant• REVERSIBLE – Activity is modulated by temperature• SIMPLE – No hot-start additives necessary • LOWCOST – 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.
OmniKlentaq®-PCRDirectlyfromBloodSamplesWhole 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.
• Increasinginstrumentthroughput• Decreasing variable (labor and consumables)
andfixed(timeoninstrument)costs
Figure3:ComparisonofOmniKlentaqvs.CommercialEnzymesinAmplificationofHepatitisBVirusfromWholeBloodSamples.A 690 bp target from Duck hepatitis B virus (DHBV) P gene was amplified from five five-fold dilutions of DNA, kit-purified 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.
OmniKlentaq®
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M 1 2 3 4 5 N 1 2 3 4 5 N M
DNA Blood BloodDNA
DNA Blood
AmpliTaqGold®
OmniKlenTaq® JumpStart®Taq
ResistanttoPCRInhibitorsinBlood
Direct PCR for Diagnostics
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TolerantofInhibitorsinWaterSamplesDetect 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 PCR-based 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.
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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 high-performance 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.
RealtimePCRofInhibitor-RichSamplesPerhaps 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.
SoilInhibitionResistantLow 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.
Figure5:OmniKlentaq®OutperformsCommercialTaqsinDirectPCRDetectionofSoil-BornMicroorganismsinCrudeSoilSamples.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.
OmniKlentaq®
FastStartTaq JumpStart®Taq OmniKlenTaq®
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FastStartTaq JumpStart®Taq OmniKlenTaq®
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RobustPCRinCrudeMilkSamples
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 Figure6.
Figure6:AmplificationofSalmonellaDNAinCrudeMilkSamples. 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.
EnzymaticsInc.100 Cummings Center Suite 407JBeverly, MA 01915 Phone (888) 927 - 7027 Fax (978) 927 - 7127 www.enzymatics.com
Enzymatics®: TheNewStandardinEnzymeProductionQuality, Service, and Value You Can Count OnEnzymatics 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 13485 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 aproteinproductsandservicespartnerthatstandsclearlyabovethecrowd,wewanttohearfromyou.
LimitationsofUse: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.
