Measuring the absorption of a sample at 260 nm is a widely used method for. 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 the overestimation of the amount of DNA and possibly to a lower DNA concentration than required for the subsequent downstream procedure. Many of these impurities can be estimated by measuring the absorption of the sample at wavelengths other than 260 nm.
High absorbance between 320nm and 340 nm is an indicator of particles in suspension. Since they normally do not influence any downstream methods the measurement at 340nm is usually subtracted from the absorbance measurement to correct the influence of particles on quantification. This subtraction is sometimes referred to as spectrum normalization.
Proteins have a higher absorption at 280 nm than at 260 nm and the ratio between the absorbances at 260 and 280 nm is a broadly accepted means of assessing protein contamination in a sample of purified DNA. The 260/280 ratio of a sample containing pure DNA with no protein contamination should be between 1.8 and 2.0. Values below 1.8 indicate contamination by protein and ratios above 2.0 indicate contamination by RNA. A low 260/280 ratio may also suggest the presence of phenol which is an additive used in some DNA purification methods.
The sensitivity of the 260/280 ratio for the detection of protein contamination is very low: a ratio of 1.75 which is only 0.05 below 1.80. This could indicate a protein content of about 50% in the sample. In this example our DNA concentration would be only half the concentration calculated by 260. Some systems offer a corrected DNA concentration based on the deviation of the samples spectrum from the theoretical spectrum of pure DNA. High amounts of protein are required for the difference to be measurable making this correction unreliable.
Proteins are not the only possible contaminant in purified DNA samples, some other common contaminants cause a relative increase in absorbance at 230 nm compared to 260 nm. The 260/230 ratio is also used to assess DNA purity and the 260/230 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 the 260/280 ratio is normally preferred as an indicator of protein contamination as it is 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. Using concentrations close to the detection limit of both the 260/280 and the 260/230 are too inaccurate to assess the DNA purity. This is due to the measured values which are very close to the detection limit of the instrument and the variability of the measurement compared to the measured values is enormous. The 280nm and 230nm values are even lower than those of 260nm and closer to the detection limit of the instrument. For example, the 260 of a sample with 2 ng/µL of pure DNA would be 0.04 and its theoretical 280 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 280 or 230 going below 0.
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. If the DNA purity in the sample is not very good DNA concentrations reported by 260 may be higher than those reported when using fluorescent dyes. Using fluorescent dyes for DNA quantification does not provide a direct estimate of the presence of contaminants which may be important to evaluate the possible effects on downstream methods. If the presence of contaminating proteins or RNA needs to be quantified to optimize the purification process, then multiple aliquots of the same sample must be taken, and each macromolecule needs to be quantified separately.
Finally, a few words on the evaluation of the integrity of nucleic acids and RNA. While the spectrum of the sample is very informative about the purity of the extracted nucleic acids 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, a 2:1 ratio is indicative for intact RNA.