Nucleotides

Thermo Fisher Scientific is one of the few primary manufacturers of nucleotides in the industry. Our nucleotides, including dNTPs, NTPs, and modified nucleotides, are synthesized from high-quality raw materials in state-of-the-art production facilities and available in a variety of formats, formulations, and volumes for convenience and flexibility. Review our offerings below for your applications like PCR, cDNA synthesis, oligo synthesis and labeling, in vitro transcription and many more.

For deoxyribonucleotide triphosphate (dNTPs), choose from our catalog of individual products (dATP, dCTP, dGTP, dTTP, and dUTP). dNTPs are available in solutions of each individual species or as a mix of all four species. Custom formulations are also available for specific requirements.

All ribonucleotide triphosphate (NTPs) formulations are designed for convenience and flexibility. Individual nucleotides (APT, CTP, GTP, and UTP) are supplied in concentrations up to 200 mM in Tris or sodium salt. Custom formulations and nucleotide mixes are also available.

dNTPs and NTPs

Table 1. Specification for dNTPs and NTPs.

  dNTPs NTPs
  dNTPs NTPs
Purity* >99% >99%
Presence of contaminating nucleotides, nucleosides and PCR inhibitors* None detected None detected
DNase, RNase, endo- and exodeoxyribonuclease** None detected None detected
Presence of human and E.coli DNA*** Negative Negative
Stability
  • 3 years at –20°C
  • >100 freeze-thaw cycles
  • 3 years at –20°C
  • >100 freeze-thaw cycles
Applications
  • qPCR, RT-qPCR
  • PCR, RT-PCR, cDNA synthesis
  • High-fidelity and long-range PCR
  • Isothermal amplification
  • DNA labeling
  • Cloning
  • Sanger sequencing and next-generation sequencing (NGS)
  • In vitro transcription
  • mRNA synthesis
  • siRNA synthesis
  • RNA amplification
  • Ligation
  • Phosphorylation
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* HPLC (High Performance Liquid Chromatography)
** Incubation of labeled DNA or RNA target with NTPs or dNTPs
*** qPCR test for Alu repeats or 23S rRNA gene
† Stability studies assessed pH, HPLC profiles, and PCR performance.

Modified nucleotides

Table 2. Modified nucleotides for in vitro transcription.

  Pseudo UTP 1-Me pseudo UTP 5-Methoxy UTP N1-Ethyl pseudo UTP 5-Methyl CTP N4-Acetyl-CTP
Purity by HPLC >99% >99% >99% >99% >99% >99%
Salt form Sodium Sodium or Tris Sodium or Tris Tris Sodium Sodium
Concentration 100 ± 3 mM 100 ± 3 mM 100 ± 3 mM 100 ± 6 mM 100 ± 3 mM 100 ± 3 mM
pH 7.7 ± 0.2 7.7 ± 0.2 7.0 ± 0.2 7.7 ± 0.2 7.0 ± 0.2 7.0 ± 0.2
Endotoxin ≤1.0 EU/ml ≤1.0 EU/ml ≤1.0 EU/ml ≤1.0 EU/ml ≤1.0 EU/ml ≤1.0 EU/ml
Free of DNase, RNase, Protease            
Animal origin free            
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Table 3. Modified nucleotides used for RNA capping.

  Cap analog
[m7G(5’)ppp(5’)G]
Anti-Reverse Cap analog (ARCA)
[3’-O-Me-m7G(5’)ppp(5’)G]
Concentration 40 mM 40 mM
Purity by HPLC >95% >95%
Endo-, exo-nuclease activity None detected None detected
RNase activity None detected None detected
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Table 4. Modified nucleotides used for labelling RNA and DNA.

  Fluorescein 12-dUTP DyLight 3 dUTP DyLight 3 CTP DyLight 5 dUTP DyLight 5 CTP DyLight DY648 dCTP Texas Red 12-dUTP
Concentration 1mM 1 mM 1 mM 1 mM 1 mM 1mM 1 mM
pH 9.0 7.5 7.6 7.5 7.6 7.0  
Purity by HPLC >95% >95% >95% >95% >95% >90% >95%
Excitation max 496 nm 550 nm 550 nm 649 nm 650 nm 653 nm 594 nm
Emission max 520 nm 570 nm 568 nm 670 nm 668 nm 674 nm 611 nm
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Ordering information

Quality

Highly sensitive diagnostic assays and therapeutic regulatory standards require the highest nucleotide quality and batch-to-batch consistency. We operate under a quality management system certified to ISO 13485 and/or ISO 9001 standards, assuring each production lot is assayed by the most stringent criteria regarding purity and functionality.

Purity testing assesses production lots for the desired species via HPLC (>99%) and monitors for the presence of nucleosidic byproducts, residual chemicals, or macromolecule contaminants that occur during production and purification processes. We use a variety of methods to assay for overall purity including HPLC, sensitive colorimetric assays, or performance in PCR (Table 5).

Table 5. Purity assessment of nucleotide solutions.

Species Detection method
Nucleosidic contaminant HPLC
Pyrophoshate Malachite Green phosphate assay
Macromolecule contaminants qPCR assay followed by gel analysis

Triphosphate purity

We manufacture nucleotides with greater than 99% triphosphate purity as assessed by HPLC chromatography (Figure 1).

Exceptional purity of NTPs
Figure 1. Exceptional purity of NTPs. HPLC analysis shows >99% triphosphate purity with negligible diphosphate and undetectable monoand tetraphosphate forms.

Nucleosidic contaminants

Trace amount of nucleosidic contaminants can significantly affect PCR performance. These include:

  • Mono- and diphosphate forms, which can decrease nucleotide incorporation
  • Di-deoxy base forms, which can terminate the amplification reaction
  • Modified nucleotides, which can decrease the sensitivity of an assay

Inorganic species

Residual chemicals used during nucleotide production or purification can interfere with PCR and are often termed “PCR inhibitors”. Pyrophosphate, a residual contaminant that can occur during production, can inhibit real-time PCR at concentrations of 0.1-0.3 mM, by interfering with fluorescence detection (Ginzinger, D.G., 2002 "Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream" Experimental Hematology 30:503-512 ). The presence of inorganic pyrophosphate is easily monitored using the Malachite Green phosphate assay which is a colorimetric method that detects formation of a complex between Malachite Green, molybdate, and free orthophosphate.

Macromolecule contaminants

The presence of macromolecules can cause false test results. These include:

  • DNase and RNase contaminants can affect cDNA synthesis resulting in false-negative results (Figure 2)
  • Nickase and protease activity can compromise the amplification template
  • Contaminating DNA from human and bacterial origins can cause false-positive results
Evaluation of ribonuclease-free Thermo Scientific dNTPs

Figure 2. Evaluation of ribonuclease-free Thermo Scientific dNTPs. Three dNTP formulations (all at 1 mM concentration) were incubated with 1 μg of RNA transcript (1 kb and 0.2 kb) for 4 hours at 37°C in 20 μL volume. The dNTP preparations are determined to be RNase free if there is no visible trace of RNA transcript degradation. M: Thermo Scientific RiboRuler Low Range RNA Ladder, ready-to-use (Cat. No. SM1833). K1: Control RNA transcript, 1 kb long. K2: Control RNA transcript, 0.2 kb long. A: With dNTP Mix, 2 mM each (Cat. No. R0241). B: With dNTP Mix, 10 mM each (Cat. No. R0191). C: With dNTP Set (Cat. No. R0181).

To complement analytical tests for quality control, each lot of Thermo Scientific nucleotides is also analyzed using a variety of functional tests including RT-qPR and in vitro transcription.

dNTPs

Samples of each lot are tested by RT-qPCR (Figure 3). The assay is performed with low template concentration, which allows detection of even minute variations in dNTP performance.

High quality of dNTPs helps ensure consistent Ct values in qPCR, especially with low template concentrations

Figure 3. High quality of dNTPs helps ensure consistent Ct values in qPCR, especially with low template concentrations. dNTPs were tested for detection of the GAPDH gene in two-step RT-qPCR using different amounts of RNA transcript (107 to 103 copies) in reverse transcription reactions followed by amplification with hot-start Taq DNA polymerase.

NTPs

Samples of each lot are tested by in vitro transcription (Figure 4).

NTP performance in high-yield in vitro transcription

Figure 4. NTP performance in high-yield in vitro transcription. RNA transcripts were generated using Thermo Scientific NTPs and NTPs from two other vendors in high-yield in vitro transcription reactions containing 1 μg of DNA template. M: Thermo Scientific RiboRuler High Range RNA Ladder, ready-to-use (Cat. No. SM1823). 1, 4, 7: 0.5 kb RNA transcripts. 2, 5, 8: 1 kb RNA transcripts. 3, 6, 9: 6 kb RNA transcripts.

Purity testing assesses production lots for the desired species via HPLC (>99%) and monitors for the presence of nucleosidic byproducts, residual chemicals, or macromolecule contaminants that occur during production and purification processes. We use a variety of methods to assay for overall purity including HPLC, sensitive colorimetric assays, or performance in PCR (Table 5).

Table 5. Purity assessment of nucleotide solutions.

Species Detection method
Nucleosidic contaminant HPLC
Pyrophoshate Malachite Green phosphate assay
Macromolecule contaminants qPCR assay followed by gel analysis

Triphosphate purity

We manufacture nucleotides with greater than 99% triphosphate purity as assessed by HPLC chromatography (Figure 1).

Exceptional purity of NTPs
Figure 1. Exceptional purity of NTPs. HPLC analysis shows >99% triphosphate purity with negligible diphosphate and undetectable monoand tetraphosphate forms.

Nucleosidic contaminants

Trace amount of nucleosidic contaminants can significantly affect PCR performance. These include:

  • Mono- and diphosphate forms, which can decrease nucleotide incorporation
  • Di-deoxy base forms, which can terminate the amplification reaction
  • Modified nucleotides, which can decrease the sensitivity of an assay

Inorganic species

Residual chemicals used during nucleotide production or purification can interfere with PCR and are often termed “PCR inhibitors”. Pyrophosphate, a residual contaminant that can occur during production, can inhibit real-time PCR at concentrations of 0.1-0.3 mM, by interfering with fluorescence detection (Ginzinger, D.G., 2002 "Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream" Experimental Hematology 30:503-512 ). The presence of inorganic pyrophosphate is easily monitored using the Malachite Green phosphate assay which is a colorimetric method that detects formation of a complex between Malachite Green, molybdate, and free orthophosphate.

Macromolecule contaminants

The presence of macromolecules can cause false test results. These include:

  • DNase and RNase contaminants can affect cDNA synthesis resulting in false-negative results (Figure 2)
  • Nickase and protease activity can compromise the amplification template
  • Contaminating DNA from human and bacterial origins can cause false-positive results
Evaluation of ribonuclease-free Thermo Scientific dNTPs

Figure 2. Evaluation of ribonuclease-free Thermo Scientific dNTPs. Three dNTP formulations (all at 1 mM concentration) were incubated with 1 μg of RNA transcript (1 kb and 0.2 kb) for 4 hours at 37°C in 20 μL volume. The dNTP preparations are determined to be RNase free if there is no visible trace of RNA transcript degradation. M: Thermo Scientific RiboRuler Low Range RNA Ladder, ready-to-use (Cat. No. SM1833). K1: Control RNA transcript, 1 kb long. K2: Control RNA transcript, 0.2 kb long. A: With dNTP Mix, 2 mM each (Cat. No. R0241). B: With dNTP Mix, 10 mM each (Cat. No. R0191). C: With dNTP Set (Cat. No. R0181).

To complement analytical tests for quality control, each lot of Thermo Scientific nucleotides is also analyzed using a variety of functional tests including RT-qPR and in vitro transcription.

dNTPs

Samples of each lot are tested by RT-qPCR (Figure 3). The assay is performed with low template concentration, which allows detection of even minute variations in dNTP performance.

High quality of dNTPs helps ensure consistent Ct values in qPCR, especially with low template concentrations

Figure 3. High quality of dNTPs helps ensure consistent Ct values in qPCR, especially with low template concentrations. dNTPs were tested for detection of the GAPDH gene in two-step RT-qPCR using different amounts of RNA transcript (107 to 103 copies) in reverse transcription reactions followed by amplification with hot-start Taq DNA polymerase.

NTPs

Samples of each lot are tested by in vitro transcription (Figure 4).

NTP performance in high-yield in vitro transcription

Figure 4. NTP performance in high-yield in vitro transcription. RNA transcripts were generated using Thermo Scientific NTPs and NTPs from two other vendors in high-yield in vitro transcription reactions containing 1 μg of DNA template. M: Thermo Scientific RiboRuler High Range RNA Ladder, ready-to-use (Cat. No. SM1823). 1, 4, 7: 0.5 kb RNA transcripts. 2, 5, 8: 1 kb RNA transcripts. 3, 6, 9: 6 kb RNA transcripts.

Custom service and flexible packaging

  • Format – any concentration, formulation, and volume; custom mixes and blends
  • Scale – microliters to liters, milligrams to kilograms
  • Packaging – labeling, finishing, and choice of vials and bottles

Need higher grade nucleotides?

We also offer TheraPure NTPs and modified nucleotides for nucleic acid therapeutics development. These high-quality nucleotides are produced with strictly controlled manufacturing processes and well-defined impurity profiles.
Contact us to learn more at NATxOEM@thermofisher.com

Support

  • Contact us – Email or call our technical application scientists for additional questions about dNTPs, NTPs, and modified nucleotides.
  • OEM and custom solutions – Contact us for tailor-made performance tested products with strict quality standard to meet your molecular assay requirements.