dNTP

[2] For dUTP: Low Copy PCR (1 kb, lambda DNA, template dilution series)

[3] Greisen et al. (1994) J. Clin. Microbiol. 32(2):335

[4] Fields et al. (2001) Toxicol. Sci. 63:107

[5] Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sn, U

The level of sophistication in PCR applications constantly reaches new highs. Performance can be negatively affected by even one poor quality reagent. That’s why we ensure the highest quality in our dNTP solutions. According to our ISO 9001:2015 certified quality management system each dNTP lot is assayed under stringent criteria for purity and functionality (see Specifications). The quality of a dNTP solution can be assessed with three criteria: Purity, Macromolecular Contaminants, and Inorganic Species.

Common contaminants in dNTP solutions are:

• Deoxyribonucleotide diphosphates – dNDPs 
• Ribonucleotides – NTPs
• Other dNTPs – e.g. deaminated or methylated dNTPs
• Other nucleosidic compounds
• Phosphates 

Of these contaminants, only the dNDPs minimally affect PCR performance. Even trace quantities of other impurities can significantly affect performance.

Figure 1: A multiplex qPCR assay verifies the absence of contaminating bacterial or human DNA in  Jena  Bioscience dNTP solutions. The 16S rRNA gene is amplified for detection of bacterial DNA. Human DNA is detected by amplification of a beta-actin gene fragment. Traces of contaminating DNA are typically detected at ct-values between 35 and 45.

Macromoleular Contaminants

The absence of human DNA and bacterial DNA in dNTP solutions is critical. These macromolecules can be present from the use of bacterial enzymes used in production, as well as human DNA from handling during production.

Since the presence of just a few copies of genomic DNA can cause false positives in PCR, we analyse each dNTP lot at the end of production to ensure they are DNA-free. (Figure 1) In addition, we test for residual enzymatic activity to ensure absence of DNases, RNases, Nicking activities, as well as proteases.

Inorganic Species

The presence of critical concentrations of inorganic species may result from contaminated raw materials and inadequate manufacturing processes. These species commonly interfere with PCR and are commonly referred to as “PCR inhibitors”. These do not  absorb  UV  light  and  are  not  detected  by  UV-detection in reverse phase HPLC. To ensure our products are free from inhibiting quantites of these species, we use Analytical Anion Chromatography, GC/FID, and ICP-MS for analyses.

[1] Standard concentration in PCR

[2] Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sn, U

Purity

To accurately assess purity, we have optimised HPLC methodology to allow for separation of similar components. Figure 2 shows a example comparison of Jena Bioscience dATP solution to fourdATP solutions from other suppliers. The peaks for dATP and dADP are labelled; other peaks present are potentially harmful to PCR performance.

Figure 2: Comparison of HPLC traces for Jena Bioscience dATP solution with four other leading suppliers. Jena Bioscience dATP contains fewer impurities – such as nucleosidic compounds which can inhibit PCR enzymes.

Due to the complexity of dNTP impurities, it is important to better illustrate purity. We first compared all four dNTPs (dATP, dCTP, dGTP, and dTTP) for each supplier. These values were then used to show the constitution of a dNTP mix for each supplier (Figure 3).

Figure 3: Data from individual solutions was used to compare representative dNTP mixes for each supplier. The final 1.0% of the purity is enlarged to highlight the differences in potentially harmful impurities between dNTP solutions from different suppliers.

Summary

• All suppliers have a dNTP purity over 99.5 %.
• Jena Bioscience has a higher quantity of dNDP – essentially harmless to PCR 
• Jena Bioscience has a significantly lower quantity of other impurities – those reducing PCR performance