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.


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.