Figure 1: Typical absorbance spectrum scan of a nucleic acid sample
Figure 2: User Interface of the NanoPhotometer® method result screen including sample quality control features (flagged sample for impurities)
Commercially available solutions such as Qubit™ contain fluorescence dyes specifically binding to dsDNA to be measured with a handheld reader. There are different types of kits available depending on the application and the desired concentration range. Fluorescence at first glance seems to be a viable path to accurate dsDNA concentrations for all kinds of samples, but there are limitations that come with this method including intense sample preparation to carry out this relatively costly and time consuming method. dsDNA quantification may be underestimated depending on the method used for the DNA extraction and in case of changing salt concentrations caused by potential dilution steps.
The following limitations should be noted when considering using a fluorescence dye based method for dsDNA concentration determinations:
Table 1. Recommended quantification methods for DNA
DNA | Relative Fluorescence | NanoPhotometer® |
Frozen | Suitable* | Recommended |
FFPE | Recommended | Overestimated |
Trizol-extracted | Underestimated | Recommended |
Dye-labeled microarray samples | Not suitable | Recommended |
Table 2. The benefit comparison of relative fluorescence and UV/Vis
Type of Detection | Relative Fluorescence | UV/VIS |
Detection of Contaminants | No | Yes |
Concentration Range | Kit dependent 0.01 – 1000 ng/µl | 1 – 16,500 ng/µl |
Consumables Needed | Yes | No |
Price Per Measurement | 0.71 – 1.18 USD | 0 USD |
Incubation Time | 2 – 5 min per sample | N/A |
Available Applications | Limited | Numerous |
References
Nakayama Y, Yamaguchi H, Einaga N, Esumi M (2016) Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions. PLoS ONE 11(3): e0150528. doi:10.1371/journal.pone.0150528Qubit™ is a registered trademark of Thermo Fisher Scientific. Implen® is not affiliated, sponsored or endorsed by Thermo Fisher Scientific or Qubit™.