DNA purity in UV spectrophotometry
As we described in the discussion on DNA quantification methods, measuring the absorption of a sample at 260 nm is a widely used method for quantifying DNA concentration. However, other macromolecules absorb at 260 nm: mainly proteins and RNA, but also substances used for purification. The contamination of the sample by substances that absorb at 260 nm leads to an overestimation of the amount of DNA and thus possibly to a lower DNA concentration than required for a subsequent downstream procedure. Many of these impurities can be estimated by measuring the absorption of the sample at wavelengths other than 260 nm.
A340
High absorbance at about 340 nm (or 320 nm) is an indicator of particles in suspension. Since they normally do not influence any downstream methods, the A340 is usually only subtracted from the absorbance measurement to correct the influence of particles on quantification. This subtraction is sometimes referred to as spectrum normalization.
A260/A280 ratio
Proteins have a higher absorption at 280 nm than at 260 nm. The ratio between the absorbances at 260 (A260) and 280 nm (A280) is broadly accepted as a means of assessing protein contamination in a sample of purified DNA. The A260/A280 ratio of a sample containing pure DNA with no protein contamination should be between 1.8 and 2.0, with values below 1.8 indicating contamination by protein, and ratios above 2.0 indicating contamination by RNA. A low A260/A280 ratio may also be indicative of the presence of phenol, an additive used in some DNA purification methods.
The sensitivity of the A260/A280 ratio for the detection of protein contamination is very low: a ratio of 1.75 - i.e. only 0.05 below 1.80 - could already indicate a protein content of about 50% in the sample. In this example, our DNA concentration would actually be only half the concentration calculated by A260. While some systems offer a corrected DNA concentration based on the deviation of the sample's spectrum from the theoretical spectrum of pure DNA, the high amounts of protein required for the difference to be measurable make this correction unreliable.
A260/A230 ratio
Proteins are not the only possible contaminant in purified DNA samples. Some common contaminants cause a relative increase in absorbance at 230 nm compared to 260 nm, and the A260/A230 ratio is hence also used to assess DNA purity. The A260/A230 ratio of pure DNA is 1.8. A lower ratio indicates contamination by phenol, EDTA, guanidine thiocyanate, Triton X-100 or carbohydrates. Protein contamination also increases this ratio, but A260/A280 ratio is normally preferred as an indicator of protein contamination, as it’s not affected by so wide a range of possible contaminants.
Limitations of UV spectrophotometry to asses DNA purity
The limit of detection of UV spectrophotometry is typically 2 ng/µL of DNA. This means that it is not possible to assess the purity of DNA samples below this concentration by using this method. Even at concentrations close to the detection limit, both the A260/A280 and the A260/A230 ratios are too inaccurate to asses the DNA purity. This is due to the fact that measured values are very close to the detection limit of the instrument, where the variability of the measurement compared to the measured values is enormous, and the A280 and A230 values are even lower than those of A260, and hence closer to the detection limit of the instrument. For example, the A260 of a sample with 2 ng/µL of pure DNA would be 0.04, and thus, its theoretical A280 would be 0.02. But an oscillation of just 0.01 would make ratios go from 4.0 (with an oscillation of -0.01 for a measured value of 0.01) to 1.33 (with an oscillation of +0.01 for a reading of 0.03). Therefore, it is not uncommon for ratios to give even negative values because of A280 or A230 going below 0.
DNA purity using fluorescent dyes
As fluorescent dyes are available for specific nucleic acid species (for example, dsDNA), contamination by proteins or other nucleic acid species has very little impact on the quantification of DNA using this method. Therefore, if DNA purity in the sample is not very good, DNA concentrations reported by A260 may be higher than those reported when using fluorescent dyes. However, DNA quantification using fluorescent dyes does not provide a direct estimate of the presence of contaminants (which may be important, for example, to evaluate the possible effects on downstream methods). If the presence of contaminating proteins or RNA needs to be quantified (for example, to optimize the purification process), then multiple aliquots of the sample have to be taken, and each macromolecule needs to be quantified separately.
Nucleic acid integrity
Finally, a few words on the evaluation of the integrity of nucleic acids and, in particular, RNA. While the spectrum of the sample is very informative about the purity of the extracted nucleic acids (whether the spectrum differs greatly from that of the pure nucleic acids and thus indicates low purity), it does not provide any information about its integrity. This is due to the absorption spectrum of free nucleotides being identical to that of nucleotides belonging to a large DNA or RNA molecule.
To assess RNA integrity, the best way is still running a gel electrophoresis and visualizing the RNA using an intercalating dye. The presence of lower molecular weight smears is indicative of RNA degradation. The relative intensities of 28S and 18S rRNAs may be used to calculate the integrity of RNA, with a 2:1 ratio is indicative for intact RNA.