Hot Start dNTP protocol to reduce non-specific amplification

How do hot-start dNTPs work?

Hot-start dNTPs are modified with a thermolabile protecting group (X) at the 3’ terminus. The presence of this modification blocks DNA polymerase nucleotide incorporation until the nucleotide protecting group is removed using a heat activation step. When standard cycling protocols are employed, a 0-10 minute initial denaturation step at 95°C allows for robust amplification. For faster thermal cycling protocols, an initial denaturation is not required. In many cases, all that is needed to successfully utilize Hot-start dNTPs in a PCR reaction is to replace the natural nucleotides with hot-start dNTPs.
Although we recommend using the CleanAmpTM, or Hot-start, dNTP Mix, which contains the modified nucleoside triphosphates of dA, dC, dG and dT, we have sometimes found that replacement of just one or two natural nucleotides with Hot-start dNTPs is enough to have the desired effect. Hot-start dNTPs are also available individually as a set (DNTPCA2 or DNTPCA10). Simply substitute the natural dNTPs with one or more of the corresponding Hot-start dNTPs.

CleanAmp dNTP

Handling

Prior to Use
Hot-start dNTPs are characteristically very similar to natural dNTPs and are stable in aqueous buffer at pH 8-10.5 for at least one year at -20°C. When stored incorrectly, the major point for degradation of both natural and Hot-start dNTPs is the triphosphate chain. Much like natural dNTPs, extra care should be taken not to expose Hot-start dNTPs to prolonged temperatures above -20°C. Exposure to ambient temperatures during shipping does not adversely affect product performance. We do not recommend exposure of the Hot-start dNTP stock solution to more than 24 TOTAL hours at room temperature or subjecting Hot-start dNTPs to more than 20 freeze-thaw cycles. If greater than ten freeze-thaw cycles are anticipated, distribute your stock solution accordingly into smaller aliquots.

Handling Guidelines

1. The CleanAmpTM dNTP Mix is shipped as a concentrated 2µmol or 10µmol solution of dATP, dCTP, dGTP and dTTP. dNTP Sets are shipped as a 10µmol solution of each individual dNTP. dNTPs can be diluted into a PCR buffer solution (pH range of most PCR buffers = 8 to 9) and refrozen at -20°C in smaller aliquots to ensure stability for at least one year. dNTPs are very stable in the stock solution in which they are delivered.

2. dNTPs can be stored for up to 15 days at 4°C as the dNTP stock solution.

3. We do not recommend storing Hot-start dNTPs at room temperature. Hot-start dNTPs should be thawed at room temperature or on ice, mixed by vortexing and pulse centrifugation and stored on ice during PCR set-up or aliquoting manipulations. Do NOT thaw Hot-start dNTPs by heating.

Product Use
CleanAmpTM dNTPs were designed as a replacement for natural nucleotides in reactions using standard thermophilic DNA polymerases such as Taq and Pfu. We have tested enzymes that employ reaction buffers which range in pH from 8 ~ 9 with good results. This wide range of compatible reaction buffers allows for a great deal of flexibility in DNA polymerase choice in PCR design. Although Hot-start dNTPs improve PCR performance when used with standard primers and a non-Hot-Start DNA polymerase, we have found a further benefit with other Hot Start reagents in some instances.

Use Guidelines

1. We have found Taq, both native and recombinant, to work well in all applications tested. Hot-start dNTPs were also shown to successfully block extension by mesophilic enzymes, such as Klenow DNA polymerase (refer to additional enzymes table).

2. PCR buffers with a pH range from 8 ~ 9 can be used for your PCR setup.

3. For standard thermal cycling protocols, we recommend 2.5 mM MgCl2, 400 µM Hot-start dNTPs and 1.25 units of Taq DNA polymerase. For improved performance, the Hot-start dNTP concentration can be increased. For every additional 0.2 mM concentration of Hot-start dNTPs, add at least an additional 1.0 mM of MgCl2.

4. Our data shows good PCR performance over a primer concentration range of 0.05 µM to 0.5 µM, finding 0.2 µM to work well in most cases.

5. Hot-start dNTPs are validated for amplicons up to 2 kb in length.

6. When using cDNA as your template, we recommend purifying the product using a commercially-available clean-up kit to remove unincorporated nucleotides. Should your protocol require the use of the cDNA product without purification, your cDNA synthesis product should be no more than 1/10th of the reaction volume of your PCR setup.

7. In multiplex reactions where four or more targets are amplified, the addition of KCl up to 100 mM final concentration will improve results. Be sure to take into account the KCl content of your PCR buffer in these optimizations.

Protocols for Taq DNA Polymerase

· Standard Thermal Cycling: 25 µL Endpoint PCR
· Fast Thermal Cycling: 25 µL Endpoint PCR
· Multiplexed: 25 µL Standard Thermal Cycling (2 to 7 targets)

1. For all components except Hot-start dNTPs and DNA polymerase, thaw the reaction components, vortex to mix, centrifuge briefly and store on ice.

2. Prepare Hot-start dNTPs:
a. Thaw at room temperature or on ice.
b. Vortex and pulse centrifuge to thoroughly mix.
c. If necessary, remove an aliquot of the stock solution and dilute with water or buffer (pH 8-10.5) to desired working concentration.

3. Prepare a mastermix containing all components except for the DNA template sample. Add each of the components as shown in table 1 (multiply amounts by the number of reactions needed) in a centrifuge tube, on ice.

Table 1:

Component Final Concentration Volume for 1 reaction
Forward Primer 20-500nM Variable
Reverse Primer 20-500nM Variable
10X PCR Buffer without MgCl2 1X 2.5µL
CleanAmpTM dNTP solution (DNTPCA1) 0.4mM 1µL
MgCl2 (25mM)
  Standard cycling 2.5mM 2.5µL
  Fast cycling 4.0mM 4µL
  Multiplexed 2.5mM 4µL
Taq DNA polymerase 5 units/µL (D4545)
  Standard cycling 0.05 units/µL 0.25µL
  Fast cycling 0.20 units/µL 1 µL
  Multiplexed 0.05-0.10 units/ µL 0.25-0.5µL
Water (W1754) q.s. to 25µL
Total Volume 25uL

*Note: D4545 includes Taq polymerase (D6677), 10X PCR buffer (P2317) and 25mM MgCl2.
**Note: If individual hot start dNTPs are desired, use DNTPCA2 or DNPTCA10.  The volume will need to be adjusted.

4. Mix the mastermix gently to protect the enzyme, by pipetting up and down. (Do not Vortex.) Pulse spin if necessary.

5. Aliquot 20 µL of mastermix into each thin-walled PCR tube.

6. To each 20 µL aliquot of mastermix, add 5 µL of the appropriate template DNA for a final reaction volume of 25 µL.

7. Pulse spin PCR tubes. Collect reaction solution at bottom of tube.

8. Place the tubes into a thermal cycler with a heated lid and perform the appropriate cycling conditions.
    a. For standard and multiplexed cycling:
        i. 95°C for 0-5 min
        ii. 30-40 cycles
            1. 95°C for 10 sec
            2. 48-60°C for 1-30 sec
            3. 72°C for 0.5-2 min
        iii. 72°C for 10 min
    b. For fast thermal cycling conditions:
        i. 98°C for 30 sec
        ii. 45 cycles
            1. 95°C for 5 sec
            2. 65°C for 5 sec
9. Analyze an aliquot of the completed reaction by agarose gel electrophoresis.

Adaptation for Real-Time PCR Applications:
The standard cycling protocol can be adapted for realtime experiments with the following alterations:
· Reactions should be incubated in a thermal cycler capable of real-time detection, where fluorescence data is collected at the completion of the annealing step of each cycle. Please contact the real-time instrument manufacturer for specific details on your setup.

· For SYBR® Green I-based detection, 30 or 300 nM passive ROX reference dye (1 mM, Agilent) and 0.15X SYBR Green I Nucleic Acid Stain (10,000X, Catalog No. S9430) should be included in the reaction.

· For SYTO® 9-based detection, 30 or 300 nM passive ROX reference dye (1 mM, Agilent) and 2 μM SYTO 9 Nucleic Acid Stain (5 mM, Invitrogen) should be included in the reaction.

For hydrolysis probe-based detection, 30 or 300 nM passive ROX reference dye (1 mM, Agilent) and 50-20 nM hydrolysis probe should be included. The optimal hydrolysis probe concentration should be determined by performing serial dilutions and identifying the concentration that provides the earliest Cq and maximal fluorescence intensity.

Troubleshooting

Observation
Probable Cause
Suggestion(s)
No amplification product or low amplicon yield
Insufficient activation of Hot-start dNTPs during thermal cycling
Increase the concentration of Hot-start dNTPs to up to 0.8 mM, adding MgCl2 to up to 4.0 mM.
Optimize the duration of the initial denaturation time to up to 10 minutes
Thermal cycling protocol is not optimized
Increase extension time. Generally extension times should be 1-2 minutes per kb of target.
Increase the number of thermal cycles in 5 cycle increments.
Optimize annealing temperature
Problem with reagents or reaction conditions
Prepare fresh reagents, including reaction buffer and dNTPs.
Verify that template is good in quality and of sufficient quantity.
Verify primer design to ensure adequate complementarity to the DNA target.
Optimize the MgCl2
 concentration (2.5 to 4.0 mM final
concentration).
Non-specific product
formation
Excessive off-target primer extension
Titrate the concentration of the primers or template DNA.
Reduce the amount of DNA polymerase.
Primer dimer formation
Reduce initial denaturation and denaturation times:
95°C for 0-5 min
[95°C for 10-20 sec, 48-60°C for 1-15 sec, 72°C for 0.5-2
min] 30-40 cycles
72°C for 10 min
Note: A zero initial denaturation time in primer/template systems
prone to primer dimer formation may cause a slight delay in Cq.

Mis-priming
Omit initial denaturation time and shorten annealing
time:
[95°C for 30 sec, 48-60°C for 1 sec,
72°C for 0.5-2 min] 30-40 cycles
72°C for 10 min

Additional enzymes validated for use with Hot-start dNTPs

We have found Taq, both native and recombinant, to work well in all applications tested.  Hot-start dNTPs were also shown to successfully block extension by mesophilic enzymes, such as Klenow DNA polymerase.

DNA Polymerase  Vendor pH  Units/µL
Taq  Sigma-Aldrich 8.0 5
Taq  Invitrogen 8.4 5
Taq New England Biolabs 8.3 5
Taq USB 8.6 5
Taq Enzymatics 8.3 5
Pfu Stratagene 8.8 2.5
Pfu (exo-) Stratagene 8.8 2.5
DyNAzymeTM Finnzymes 8.8 2
Deep VentR TM (exo-) New England Biolabs 8.8 2
Tth USB 8.6 5
Tfi Invitrogen 8.4 5
EconoTaq® Lucigen 9 5
Phusion® Finnzymes - 2

Standard Thermal Cycling Conditions for Other DNA Polymerases

Taq DNA Polymerase from Thermus aquaticus (Sigma-Aldrich – D4545) - 25 µL reaction
· Reaction Buffer: 10X PCR buffer without MgCl2
· Supplement with an additional 2.5 mM MgCl2 (2.5 mM final concentration)
· Taq DNA pol Stock (5 U/μL); amount per reaction = 1.25 U (0.25 μL)

Taq DNA polymerase (Invitrogen) - 25 µL reaction
· Reaction Buffer: 10X PCR buffer
· Supplement with an additional 2.5 mM MgCl2 (2.5 mM final concentration)
· Taq DNA pol Stock (5 U/μL); amount per reaction = 1.25 U (0.25 μL)

Taq DNA polymerase (New England Biolabs) - 25 µL reaction
· Reaction Buffer: 10X Standard Taq Reaction Buffer
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Taq DNA pol Stock (5 U/μL); amount per reaction = 1.25 U (0.25 μL)

Taq DNA polymerase (USB) - 25 µL reaction
· Reaction Buffer: 10X PCR Reaction Buffer
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Taq DNA pol Stock (5 U/μL); amount per reaction = 1.25 U (0.25 μL)

Taq DNA polymerase (Enzymatics) - 25 µL reaction
· Reaction Buffer: 10X PCR Reaction Buffer I
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Taq DNA pol Stock (5 U/μL); amount per reaction = 1.0 U (0.2 μL)

Pfu (exo+) and (exo-) DNA polymerase - 100 µL reaction
· Reaction Buffer: 10X Cloned Pfu buffer
· DNA polymerase Stock:
· Cloned Pfu (exo+) DNA pol (2.5 U/µL); amount per reaction = 2.5 U (1.0 µL)
· Cloned Pfu (exo-) DNA pol (2.5 U/µL); amount per reaction = 2.5 U (1.0 µL)

DyNAzyme™ II DNA polymerase - 50 µL reaction
· Reaction Buffer: 10X Optimized DyNAzymeTM Buffer
· DyNAzymeTM II DNA pol Stock (2 U/µL); amount per reaction = 2 U (1.0 µL)

Deep VentR™ (exo-) DNA polymerase - 100 µL reaction
· Reaction Buffer: 10X ThermoPol buffer
· Deep VentR™ (exo-) DNA pol Stock (2 U/µL); amount per reaction = 1.25 U (1.0 µL)

Tth DNA polymerase - 25 µL reaction
· Reaction Buffer: 10X PCR buffer
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Tth DNA pol Stock (5 U/µL); amount per reaction = 5.0 U (1 µL)

Tfi DNA polymerase - 25 µL reaction
· Reaction Buffer: 5X Tfi PCR Reaction Buffer
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Tfi DNA pol Stock (5 U/µL); amount per reaction = 5 U (1 µL)

EconoTaqTM DNA polymerase - 50 µL reaction
· Reaction Buffer: 10X PCR Reaction Buffer
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· EconoTaqTM Stock (5 U/µL); amount per reaction = 1 U (0.2 µL)

Phusion® High-Fidelity DNA polymerase - 50 µL reaction
· Supplement with an additional 1.0 mM MgCl2 (2.5 mM final concentration)
· Stock (2 U/µL); amount per reaction = 1 U (0.5 µL)

CleanAmp™ dNTPs Frequently Asked Questions

What are CleanAmp™ dNTPs and how do they work?
CleanAmp™ dNTPs, hereinafter called Hot-Start dNTPs, contain thermolabile modification groups that allow for a dNTP-mediated Hot Start activation approach in PCR. The introduction of temperature sensitive protecting groups onto the 3’-hydroxyl of a dNTP blocks primer extension at the less stringent, lower temperatures of PCR reaction preparation. When the reaction is heated to the elevated temperatures of PCR, the protecting group is removed to form the corresponding standard dNTP, which is now a suitable DNA polymerase substrate.

Why use Hot-Start dNTPs versus unmodified dNTPs?
Hot-Start dNTPs are able to decrease the formation of primer dimer products while increasing the specificity of the desired product better than unmodified dNTPs. Use of Hot-Start dNTPs also reduces data fluctuation, giving a more consistent PCR result.

How stable are Hot-Start dNTPs?
Hot-Start dNTPs are most stable in the shipped mix, diluted in PCR buffer (8-9 pH). In these conditions, they are stable for at least one year at –20°C. Exposure to higher temperatures during shipment does not pose performance risks. Avoid repeated freeze/thaw cycles and exposure to room temperature for more that 24 total hours. Upon first use, it is recommended to aliquot samples into single use portions.

What are the benefits of Hot-Start dNTPs?
Hot-Start dNTPs improve PCR specificity, make PCR reactions cleaner, eliminate or reduceoff-target amplicon formation, and are compatible with both Hot Start and non-Hot Start DNA polymerases that employ different buffer compositions (pH 7.5-9, 25°C). Hot-Start dNTPs are water soluble, stable for at least 1 year when frozen at –20°C and are inexpensive compared to other Hot Start technologies.

What does the Hot-Start dNTP Mix consist of?
It contains the modified nucleoside triphosphates of dA, dC, dG, and dT. Hot-Start dNTPs are available in a mix or individually as a set. We have found that sometimes the replacement of just one or two of the natural nucleotides with Hot-Start dNTPs is enough to have the desired effect.

How should I store Hot-Start dNTPs?
For best performance, we recommend distributing your stock solution into smaller aliquots that are sufficient for one week of work. To avoid prolonged exposure of the Hot-Start dNTP Mix stock solution to room temperature, store stock nucleotide solutions in the freezer at -20°C. We recommend not subjecting the Hot-Start dNTPs to more than 20 freeze-thaw cycles.

How should I handle Hot-Start dNTPs?
For handling stock solution, thaw for 5-15 minutes at room temperature. Do not thaw by heating. The unused portion should be returned to the freezer as quickly as possible. We recommend aliquotting stock solutions into several tubes to prevent extended room temperature exposure over many uses.

How long is the initial denaturation time for Hot-Start dNTPs?
When standard cycling protocols are employed, a 2-5 minute initial denaturation step at 95°C allows for robust amplification. Recently we have found that initial denaturation times as short as 30 seconds can be used without any effect on PCR efficiency.

How versatile are Hot-Start dNTPs?
Hot-Start dNTPs can be used with a broad selection of DNA polymerases includingTaq,Pfu,Pfu(exo-), Dynazyme™,Deep VentR™,TthandTfi. When using Hot-Start dNTPs with these polymerases, all were able to produce the desired amplicon.

Will Hot-Start dNTPs activate at 60°C?
We are currently testing the kinetics of the deprotection of Hot-Start dNTPs at lower temperatures such as 60°C. Both the temperature and the pH of the PCR reaction affect the deprotection rate of the Hot-Start dNTPs where increased temperature and acidic conditions accelerate deprotection.
Our studies of deprotection at 55°C in 1X PCR buffer (pH 8.3) indicate that 40% of the dNTPs are deprotected after 2 hrs. Although deprotection is slower than at the elevated temperatures of PCR (95°C), adjustments to your reaction conditions, such as higher dNTP concentrations will likely allow for success.
Our Hot-Start Primers contain a different modification, but provide Hot Start activation using the same principle. At lower temperatures the rate of deprotection is slower, with a lower concentration of activated primers. To counter act the slower rate, we recommend using a higher concentration of primers.

Do Hot-Start dNTPs form a modified amplicon?
No, Hot-Start dNTPs are reverted back to the corresponding standard dNTP prior to being incorporated into the amplicon.The 3’-hydroxyl modification blocks enzyme incorporation until it is removed during heat activation.After heat activation, the enzyme efficiently incorporates the corresponding standard dNTP.

Materials

     
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