A260/A280 ratio is a standard procedure that is often used to evaluate the purity of nucleic acid samples.
Initially described by Christian and Warburg, this process was developed to determine the purity of protein even when contamination is present in nucleic acid. Today, this procedure is being used to determine the purity of samples containing nucleic acid . The A260/A280 ratio is based on the Beer-Lambert Law:
OD = eCb (1)
Where optical density (OD) refers to the product of extinction coefficient (e), the sample concentration C) and the optical pathlength (b).
A 1 cm optical pathlength is often utilized in spectrophotometers. As a result, one can overlook the pathlength and the extinction coefficients can be elucidated as an absorbance value at a particular concentration, as shown the following equation.
e = OD/C (2)
Standard extinction coefficients, which are often accepted for 1 mg/ml solutions of nucleic acid at 280 nm and 260 nm, are 10 and 20, in that order. Likewise, the extinction coefficient values for proteins at 280 nm and 260 nm at 1 mg/ml concentration are 1.00 and 0.57, respectively.
Therefore, in the case of nucleic acid samples, a higher absorbance would be anticipated at 260 nm than at 280 nm, while in the case of a protein sample, it would be the inverse.
Using these extinction coefficients, pure nucleic acids would have an A260/A280 ratio of 2.0, while an A260/A280 ratio of 0.57 would be exhibited by the protein sample.
Samples containing a combination of DNA and protein would be influenced by both macromolecules. In order to predict the hypothetical A260/A280 ratio for samples containing a mixture of nucleic acid and DNA, the following formula can be used:
Where %N and %P refer to the percentage of nucleic acid and protein, respectively and n and p subscripts indicate the extinction coefficients of nucleic acid and protein.
Materials and Methods
For the experiment, E. coli bacteria strain DH5 a, containing the pUC19 plasmid, were cultured in Luria broth (LB) media at 37°C. Alkaline lysis and cesium chloride gradient banding processes, as illustrated by Maniatis et al., were used to separate and purify the plasmid DNA .
Eco RI was used to digest the purified genomic herring sperm DNA. This was followed by organic phenol/chloroform/isoamylalcohol or PCI extraction, ethanol precipitation, and then rehydration at a final concentration of 400 µg/ml.
Purified bovine serum albumin (BSA) fraction V, Sigma catalogue number A-2153, was dissolved in distilled water at 400µg/ml concentration and then filter sterilized. A Lambda 3B spectrophotometer (Perkin Elmer) was used to make spectrophotometric measurements with 1 nm band-pass setting.
Using matched Hellma quartz 1 cm cuvettes, the samples were blanked on water. BioTek’s PowerWave scanning microplate spectrophotometer was used to make microplate measurements, using Costar UV transparent microplates, catalogue number 3635. Samples were blanked at each wavelength by pre-reading the plate at both wavelengths and subtracting the empty plate absorbance.
Figure 1 data shows the peak absorbance of pure DNA and protein solutions, as well as a mixture of the two macromolecules. Each moiety demonstrates overlapping, but discernible peaks, with the peak in absorbance for DNA at 257 nm and for BSA protein at 277 nm.
The protein sample also demonstrates a very high value below 240 nm that rapidly declines by 245 nm and most likely represents the absorbance of the peptide bonds in protein.
A 10:1(w/w) mixture DNA:protein results in a peak absorbance of 259 nm and an absorbance profile very similar in shape as that demonstrated by pure DNA with a small increase at wavelengths below 240 nm and represents a sum of the two absorbance patterns of the macromolecules. The absorbance of all three samples falls to near zero above 300 nm (data not shown)
Figure 1. Absorbance profiles of DNA and proteins samples from 240 to 290 nm. The absorbance of purified plasmid DNA (80mg/ml); 3mg/ml aqueous bovine serum albumin (BSA) solution; or a 10:1 (w/w) DNA to protein mixture in aqueous solution was determined in 1nm increments from 240nm to 290nm using a Perkin Elmer Lambda 3B spectrophotometer. Similar measurements were made using a 3mg/ml aqueous bovine serum albumin (BSA) solution.
It is possible to determine the 'A280 ratio' profile,, by dividing the normalized absorbance at multiple wavelengths by the value acquired at 280nm. After performing this calculation and plotting for pure DNA, a curve with a peak at 260 nm wavelength is produced, as shown in Figure 2.
Similar to the extinction coefficients value expected for nucleic acids, the extinction coefficient value at this peak is 1.99, which is near to the hypothetically predicted value of 2.0.
Similarly, protein only samples show a peak at 280 nm, indicating the highest absorbance of proteins at this specific wavelength. The sample comprising the DNA/protein mixture demonstrates a profile that is very similar in shape as that shown by pure DNA. However, this sample has relatively lower values, in spite of having matching quantities of nucleic acid in both samples.
Figure 2. A280 ratio of samples containing DNA and/or protein. Absorbance measurements were made on samples containing either DNA; or BSA protein; or a mixture of both at wavelengths from 240nm to 290nm. A280 ratio measurements were then calculated by dividing the absorbance determination at each wavelength by the A280 determination for that sample.
When measuring the A260/A280 ratio for different mixtures of protein and DNA, it can be seen that the ratio is relatively insensitive to the addition of protein to the pure nucleic acid sample.
As demonstrated in Figure 3 as increasing percentages of protein are measured little change is seen in the A260/A280 ratio until the percentage of protein is approximately 75%.
Figure 3. Comparison of theoretical A260/A280 ratios with those determined using the PowerWave scanning microplate spectrophotometer. The absorbance of various mixtures of DNA and protein were determined at 260nm and 280nm using a BioTek Instruments PowerWave scanning microplate reader. Subsequently the A260/A280 ratios were determined for each mixture and compared to the theoretical value calculated from the extinction coefficients. Filled circles indicates theoretical ratios calculated using equation 3, while filled boxed denote experimentally determined ratios.
However, even when equal amounts of protein and nucleic acid,by weight, are determined, a 1.75 ratio is still returned. Moreover, A260/A280 ratios of 0.64 and 1.92 were observed in protein or DNA only samples, respectively.
It should be noted that the A260/A280 ratio is only an indication of purity [2, 3] rather than a precise answer. Preparations of pure RNA and DNA have expected A260/A280 ratios of greater than 2.0 and greater than 1.8, respectively and are built on nucleic acids’ extinction coefficients at 280nm and 260nm.
While changes do not considerably affect the A260/A280 ratio, making it probably insignificant when mixtures of protein and DNA analyzed at an experimental level, the A260/A280 ratio becomes important when purifying nucleic acids from blood or tissue.
Tissue samples and to a lesser extent whole cells have a protein content that greatly exceeds that of nucleic acid on a weight basis and purification of samples to a A260/A280 ratio represents an enrichment of nucleic acid that could be as much as 1 million fold.
A260/A280 ratios can be affected by a number of factors. The 260 nm measurements are made very near the peak of the absorbance spectrum for nucleic acids, while the 280 nm measurement is located in a portion of the spectrum that has a very steep slope.
As a result, very small differences in the wavelength in and around 280 nm will effect greater changes in the A260/A280 ratio than small differences at 260 nm.
Thus, different types of instruments will lead to slightly varied A260/A280 ratios on the same given solution owing to inconsistency of wavelength precision between the instruments.
Individual instruments, however, should give consistent results. Concentration can also affect the results, as dilute samples will have very little difference between the absorbance at 260 nm and that at 280 nm.
With very small differences, the detection limit and resolution of the instrument measurements begin to become much more significant. The type(s) of protein present in a mixture of DNA and protein can also affect the A260/A280 ratio determination.
Absorbance in the UV range of proteins is primarily the result of aromatic ring structures. Proteins are composed of 22 different amino acids of which only three contain aromatic side chains. Therefore, amino acids sequence in proteins could have a major effect on the protein’s ability to absorb light at 280 nm.
A protein with a very high content of amino acids with aromatic side chains would in turn have a higher extinction coefficient than a protein with very few.
For example, BSA has an extinction coefficient value of 0.7 for a 1 mg/ml solution at 280 nm, while streptavidin with an extinction coefficient of 3.4 absorbs relatively more amount of light at 280 nm at the same level of concentration.
Historically, traditional spectrophotometers were used to determine the A260/A280 ratio, but such an approach involves the use of two or four matched cuvettes to carry out the analysis, which is, at best a low throughput method.
If the PowerWave scanning microplate reader is used for this analysis, the A260/A280 ratio can be quickly carried out on 96 samples, thus resulting in higher throughput and better productivity.
Produced from materials originally authored by Paul G. Held, Ph. D. Senior Scientist & Applications Lab Manager, BioTek.
- Warburg, O. and W. Christian (1942) Isolation and crystallization of enolase. Biochem. Z. 310:384-421.
- Glasel, J.A. (1995) Validity of Nucleic Acid Purities Monitored by A260/A280 Absorbance Ratios, Biotechniques 18:62-63.
- Maniatis T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
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