Quantification of nucleic acids

Ads

Quantification of nucleic acids

Quantification of nucleic acids is commonly used in molecular biology to determine the concentrations of DNA or RNA present in a mixture, as subsequent reactions or protocols using a nucleic acid sample often require particular amounts for optimum performance. There exist several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.

Spectrophotometric quantification

Because DNA and RNA absorb ultraviolet light, with an absorption peak at 260nm wavelength, spectrophotometers are commonly used to determine the concentration of DNA in a solution. Inside 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 and RNA it is 0.027 (μg/ml)^-1 cm^-1 and for short single-stranded oligonucleotides it is dependent on the length and base composition. 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. A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.

Sample purity

It is common for nucleic acid samples to be contaminated with other molecules (eg, protein, phenol, and other organic compounds). Because these molecules have their own characteristic absorption spectra, the absorption at other wavelengths is often compared to 260nm absorption in order to assess sample purity. In addition, some contaminants (notably phenol) can significantly contribute to an error in concentration estimation as they also absorb strongly at 260nm.

Protein contamination and the 260:280 ratio

The ratio of absorptions at 260nm vs 280nm is commonly used to assess the purity of DNA with respect to protein contamination, since protein (in particular, the aromatic amino acids) tends to absorb at 280nm. The method dates back to 1942, when Warburg and Christian showed that the ratio is a good indicator of nucleic acid contamination in protein preparations. Unfortunately, the reverse is not true -- it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio.

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 of 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.

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 270nm and a 260:280 ratio of 2. Nucleic acid preparitions uncontaminated by phenol should have a 260:270 ratio of around 1.2. Contamination by phenol can significantly contribute to overestimation of DNA concentration.
Absorption at 230nm can be caused by contamination by phenolate ion, thiocyanates, and other organic compounds. For a pure nucleic acid sample, the 260:230 ratio should be around 2.
Absorption at 330nm 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 (eg. 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.