Dear Chun-Nan,
The CleanAmp™ dNTPs have a half life of 5 minutes at 95°C in PCR buffer. At 92°C the deprotection rate will be slightly slower however this temperature should allow deprotection of enough of the CleanAmp™ dNTPs to work in your PCR. If needed, you could extend the time of the initial denaturation. Please contact us if you have any additional questions.
Best Regards,
Sabrina
No, CleanAmp™ 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.
We are currently testing the kinetics of the deprotection of CleanAmp™ dNTPs at lower temperatures such as 60°C. Both the temperature and the pH of the PCR reaction affect the deprotection rate of the CleanAmp™ 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 CleanAmp™ 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.
CleanAmp™ dNTPs can be used with a broad selection of DNA polymerases including Taq, Pfu, Pfu (exo-),Dynazyme™, Deep VentR™, Tth and Tfi. When using CleanAmp™ dNTPs with these polymerases, all were able to produce the desired amplicon.
Yes, please see the CleanAmp™ dNTPs Product Manual.
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.
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.
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 CleanAmp™ dNTP Mix stock solution to room temperature, store stock nucleotide solutions in the freezer at -20°C. We recommend not subjecting the CleanAmp™ dNTPs to more than 20 freeze-thaw cycles.
It contains the modified nucleoside triphosphates of dA, dC, dG, and dT. We currently offer CleanAmp™ dNTPs in a mix but the CleanAmp™ dNTPs are also available individually upon request. We have found that sometimes the replacement of just one or two of the natural nucleotides with CleanAmp™ dNTPs is enough to have the desired effect.
CleanAmp™ dNTPs improve PCR specificity, make PCR reactions cleaner, eliminate or reduce off-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). CleanAmp™ dNTPs are water soluble, stable for at least 1 year when frozen at –20°C and are inexpensive compared to other Hot Start technologies.
CleanAmp™ 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.
CleanAmp™ 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 CleanAmp™ dNTPs also reduces data fluctuation, giving a more consistent PCR result.
CleanAmp™ 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.