I want to conduct 3D PCR as desribed by Suspene et al. I have ordered DIT and dDTP from your company. Is 2-amino-2′-dATP the dDTP that Suspene is describing? And is Pfu polymerase able to incorporate these nucleotides? -Bernadette

Dear Bernadette,

Thank you for your inquiry. Yes, 2-amino-2′-dATP is the dDTP that Suspene describes. We do not know for sure whether pfu polymerase is able to incorporate these nucleotides but it probably doesn’t. We recommend trying NEB’s Therminator.

Please let me know if you have any additional questions.

Regards,
Brea

My NTP shipment arrived without dry ice. Should I be concerned?

We ship our triphosphates on dry ice as a precaution. They are stable at room temperature, and even higher temperatures, for short periods of time. The chance of degradation during transit is very low. We recommend analyzing the material by AX-HPLC and then testing it in your application, provided the cost of the assay is not prohibitive. Contact us to request our analysis method.

What base modifications can be incorporated into my custom mRNA or long RNA?

TriLink offers a variety of modified rNTPs suitable for in vitro transcription. We have assessed transcription efficiency based on final product formation of a 1.9 kb transcript with the following triphosphates at 100% substitution using T7 polymerase.

NTP Transcription Efficiency
5-Aminoallyl-CTP (N-1065) +++
2-Amino-ATP (N-1001) +++
5-Br-UTP (N-1054) +
5-Carboxy-CTP (N-1084) ++
5-Carboxy-UTP (N-1091) n/a
5-Carboxymethylester-UTP (N-1096) ++
7-Deaza-ATP (N-1061) ++
5-Formyl-CTP (N-1085) +++
5-Formyl-UTP (N-1090) +
5-Hydroxy-CTP (N-1089) ++
5-Hydroxy-UTP (N-1092) ++
5-Hydroxymethyl-CTP (N-1087) ++
5-Hydroxymethyl-UTP (N-1086) +++
5-Iodo-UTP (N-1012) +
5-Methoxy-CTP (N-1094) ++
5-Methoxy-UTP (N-1093) ++
N6-Methyl-Amino-ATP (N-1083) ++
N6-Methyl-ATP (N-1013) +
5-Methyl-CTP (N-1014) +++
Pseudo-UTP (N-1019) +++
Thieno-CTP (N-1097) n/a
Thieno-GTP (N-1088) ++
1-Thio-ATP (N-8005) +
2-Thio-UTP (N-1032) +++

+++ = Greater than 75% efficiency compared to unmodified NTP
++ = Between 25-75% efficiency compared to unmodified NTP
+ = Less than 25% efficiency compared to unmodified NTP
n/a = No significant product formed with 100% substitution

In all cases, transcription was carried out at 37°C.

Which of your modified nucleoside triphosphates can be incorporated into DNA molecules through PCR?

Aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 7-deaza-dGTP, and 7-deaza-dATP, 5-Methyl-dCTP, 5-Iodo-dUTP, 5-Bromo-dUTP, 5-Fluoro-dUTP, N4-methyl-dCTP, 5-propynyl-dUTP and 5-propynyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and alpha-thio-dNTPs can be incorporated through PCR. As not all of these analogs will incorporate with similar efficiencies in PCR; some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.

Which enzymes work with which modified nucleotides?

There is very little known about many of the triphosphates we sell. Please see our table of Enzymatic Activity of Selective NTPs, or our NTP bibliography in our website and catalog to see if there is a publication regarding your question. If not, our stock answer is often tongue in cheek: buy the compound, be the first to do the research, publish the paper and then we will both know. Seriously, we are attempting through both in-house and collaborative efforts to learn more about potential applications of our compounds, but this is a long term effort. Please contact us to see if there is any news about your particular application.

How do you make sure that no salts are left in your preparations after purification and precipitation?

We QC our products by 31P NMR which reveals inorganic phosphate salt. This is usually the salt of most concern for enzymologists since it is an inhibitor of many polymerases. For other salts, the lambda max and epsilon values of the modified nucleoside are used to make sure the optical density matches the actual dry weight. The concentration of NTP in the final solution is determined spectrally. This spectra is not affected by the salt form of the triphosphate, assuring we deliver accurate quantities of our NTPs, based on the molecular weight of the free acid form.

What are the likely contaminants when preparing a triphosphate?

The most common contaminants are the mono- and diphosphate forms of the nucleotide. Our purification procedures are designed to minimize the amount of other phosphates present, but since they result from hydrolysis that occurs at some finite rate with all nucleotides, they are generally present in the 0.5-3% range as detected by anion exchange HPLC. We routinely analyze older lots to identify these species and repurify the NTP if necessary to ensure it meets our purity specification (>90% for catalog products) before shipping. Two other potential contaminants are salts and inorganic phosphate. Salts are removed during the reverse phase HPLC and precipitation steps. Inorganic phosphate can be particularly problematic in some applications, therefore we take special measures to remove as much as possible. Every lot of NTP we manufacture is analyzed for the presence of inorganic phosphate using phosphorous NMR.

How do you prepare your nucleotides?

All of our nucleotides are prepared chemically using a variety of methods, several of which are proprietary to TriLink. We use a multi-step purification process that includes two DEAE columns and a reverse phase HPLC. Finally, the compounds are precipitated to remove any remaining salts and analyzed for quality. They are stored as an aqueous solution at -70°C.

My NTP arrived thawed. Is it ok?

Although NTPs are stable at room temperature for over a week, we ship them on ice to ensure quality in the event of a delay in transportation. We are still confident in the integrity of our NTPs if they have arrived thawed. However, if your ice has disappeared when your NTP arrives and you have concerns, please contact us immediately.

How should I store my NTP and how long will it last?

Most NTPs should be stable for several years when stored properly at -20oC. We recommend aliquotting your NTP into multiple, single-use tubes upon first thawing to avoid repeated freeze/thaw cycles, which can lead to significant physical degradation. This is especially important for NTPs with amino modifications, which are less stable in solution than other NTPs.

What type of QC do you run on your triphosphates?

After purifying an NTP by anion exchange and reverse phase HPLC, the compound is precipitated and dissolved in water, an analytical scale anion exchange HPLC is run to verify each compound is >90% triphosphate. In addition, 31P NMR, Mass Spec and UV analyses are done to ensure the triphosphate has little or no inorganic phosphate. We then match the calculated molecular weight and confirm the concentration of the final solution.