In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance. There are several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.
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Nucleic acids absorb ultraviolet light in a specific pattern. In a spectrophotometer, a sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample.
Using the Beer Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule. At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/ml)-1 cm-1, for single-stranded DNA it is 0.027 (μg/ml)-1 cm-1, for single-stranded RNA it is 0.025 (μg/ml)-1 cm-1 and for short single-stranded oligonucleotides it is dependent on the length and base composition (estimation 0.030 (μg/ml)-1 cm-1). Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an OD of at least 2.[1] A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.[2]
| Nucleic Acid | Concentration (μg/ml) for 1 A260 unit |
|---|---|
| dsDNA | 50 |
| ssDNA | 37 |
| ssRNA | 40 |
The optical density of samples measured with 10 mm path length standard cuvettes simply has to be multiplied by the conversion factor to determine the concentration of the sample. Example, a dsDNA sample with an OD of 0.9 corresponds to a sample concentration of 45 µg/ml.
Multiple biological applications (e.g. DNA microarray experiment, array CGH, qPCR) imply quantitative and qualitative nucleic acid analysis with minimal sample volumes. Specialized NanoPhotometer[3] offer the possibility to determine sample concentrations cuveteless with submicroliter volumes (starting with 0.3 µl). In addition, due to the reduction of the optical pathlength samples are diluted automatically in comparison to standard cuvette measurements. The respective virtual dilution factors are considered by the software of the instrument. Because the measurements are processed with undiluted samples, the reproducibility of the results is very high. And if desired, samples can be retrieved after the measurement for further processing.
It is common for nucleic acid samples to be contaminated with other molecules (i.e. proteins, organic compounds, other). The ratio of the absorbance at 260 and 280nm (A260/280) is used to assess the purity of nucleic acids. For pure DNA, A260/280 is ~1.8 and for pure RNA A260/280 is ~2.
The ratio of absorptions at 260nm vs 280nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280nm.[1][4] The reverse, however, is not true — it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio in a nucleic acid solution.[1][5]
260:280 ratio has high sensitivity for nucleic acid contamination in protein:
| % protein | % nucleic acid | 260:280 ratio |
|---|---|---|
| 100 | 0 | 0.57 |
| 95 | 5 | 1.06 |
| 90 | 10 | 1.32 |
| 70 | 30 | 1.73 |
260:280 ratio lacks sensitivity for protein contamination in nucleic acids:
| % nucleic acid | % protein | 260:280 ratio |
|---|---|---|
| 100 | 0 | 2.00 |
| 95 | 5 | 1.99 |
| 90 | 10 | 1.98 |
| 70 | 30 | 1.94 |
This difference is due to the much higher extinction coefficient nucleic acids have at 260nm and 280nm, compared to that of proteins. Because of this, even for relatively high concentrations of protein, the protein contributes relatively little to the 260 and 280 absorbance. While the protein contamination cannot be reliably assessed with a 260:280 ratio, this also means that it contributes little error to DNA quantity estimation.
An alternative way to assess DNA concentration is to use measure the fluorescence intensity of dyes that bind to nucleic acids and selectively fluoresce when bound (e.g. Ethidium bromide). This method is useful for cases where concentration is too low to accurately assess with spectrophotometry and in cases where contaminants absorbing at 260nm make accurate quantitation by that method impossible.
There are two main ways to approach this. "Spotting" involves placing a sample directly onto an agarose gel or plastic wrap. The fluorescent dye is either present in the agarose gel, or is added in appropriate concentrations to the samples on the plastic film. A set of samples with known concentrations are spotted alongside the sample. The concentration of the unknown sample is then estimated by comparison with the fluorescence of these known concentrations. Alternatively, one may run the sample through an agarose or polyacrylamide gel, alongside some samples of known concentration. As with the spot test, concentration is estimated through comparison of fluorescent intensity with the known samples.[1]
If the sample volumes are large enough to use microplates or cuvettes, the dye-loaded samples can also be quantified with a fluorescence photometer.
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