Methods and Concepts in the Life Sciences/Nucleic Acid Quantitation
Nucleic acid quantitation
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. A method for the quantitation of specific nucleic acids is real-time PCR.
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. Thus, an optical density (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. A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.
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.
Protein contamination and the 260:280 ratio
The ratio of absorptions at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm. 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.
260:280 ratio has high sensitivity for nucleic acid contamination in protein:
|% protein||% nucleic acid||260:280 ratio|
260:280 ratio lacks sensitivity for protein contamination in nucleic acids (table shown for RNA, 100% DNA is approximately 1.8):
|% nucleic acid||% protein||260:280 ratio|
This difference is due to the much higher extinction coefficient nucleic acids have at 260 nm and 280 nm, 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.
Other common contaminants
- Contamination by phenol, which is commonly used in nucleic acid purification, can significantly throw off quantification estimates. Phenol absorbs with a peak at 270 nm and a A260/280 of 1.2. Nucleic acid preparations uncontaminated by phenol should have a A260/280 of around 2. Contamination by phenol can significantly contribute to overestimation of DNA concentration.
- Absorption at 230 nm can be caused by contamination by phenolate ion, thiocyanates, and other organic compounds. For a pure RNA sample, the A230:260:280 should be around 1:2:1, and for a pure DNA sample, the A230:260:280 should be around 1:1.8:1.
- Absorption at 330 nm and higher indicates particulates contaminating the solution, causing scattering of light in the visible range. The value in a pure nucleic acid sample should be zero.
- Negative values could result if an incorrect solution was used as blank. Alternatively, these values could arise due to fluorescence of a dye in the solution.
Quantification using fluorescent dyes
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.
If the sample volumes are large enough to use microplates or cuvettes, the dye-loaded samples can also be quantified with a fluorescence photometer.