Deoxyribonucleic acid (DNA) is the fundamental instruction manual for all living organisms. Understanding how much DNA is present in a sample is a primary step in virtually every molecular biology experiment. Scientists use spectrophotometry to measure the concentration and purity of DNA in a solution. This method works by shining light through the sample and measuring how much is absorbed, relying on the intrinsic chemical properties of the DNA molecule. The most intense absorption of light by DNA occurs in the ultraviolet (UV) region of the spectrum, with a precisely defined peak at the 260 nanometer (nm) wavelength.
Why DNA Absorbs Ultraviolet Light
The reason DNA strongly absorbs UV light at 260 nm lies in the chemical structure of its building blocks, specifically the nitrogenous bases. These bases, which include adenine, guanine, cytosine, and thymine, are all aromatic compounds. Aromatic molecules contain ring structures with alternating single and double bonds, creating a system of delocalized electrons.
These systems of conjugated double bonds act as chromophores, the part of a molecule responsible for absorbing light. When a photon of UV light at the 260 nm wavelength hits these electron systems, the energy is absorbed, causing an electron to jump to a higher energy state. This molecular interaction is the physical basis for the observed absorption peak. Neither the deoxyribose sugar nor the phosphate backbone contribute significantly to the UV absorption at this wavelength, as they lack these aromatic ring structures.
The overall 260 nm peak for double-stranded DNA is the combined average of the individual absorption maxima of the four different bases. While each base has a slightly different maximum absorption wavelength—for example, guanine peaks at 249 nm and adenine at 260 nm—the collective spectrum results in a sharp, measurable maximum near 260 nm.
Quantifying DNA Concentration
The primary practical application of the 260 nm absorbance measurement is the precise quantification of DNA concentration in a solution. This process is governed by the Beer-Lambert Law, which states that the absorbance of light is directly proportional to the concentration of the absorbing substance and the distance the light travels through the solution. In a typical laboratory spectrophotometer, the light path length is standardized to one centimeter (cm).
By measuring the absorbance at 260 nm (A260), scientists calculate the nucleic acid concentration using established conversion factors. For double-stranded DNA (dsDNA), an A260 reading of 1.0, measured in a 1 cm cuvette, corresponds to a concentration of 50 micrograms per milliliter (\(\mu\)g/mL).
For single-stranded DNA (ssDNA) or RNA, the conversion factors are slightly different, typically set at 33 \(\mu\)g/mL and 40 \(\mu\)g/mL, respectively, per 1.0 A260 unit. This difference is due to the varying spatial arrangements of the bases, which affects their light absorption efficiency. This spectrophotometric method measures the concentration of all nucleic acids present, meaning the A260 reading reflects the total concentration of both DNA and RNA if both are in the sample.
Highly concentrated samples must often be diluted so the A260 reading falls within the instrument’s linear range, typically between 0.1 and 1.0 absorbance units, to ensure accurate results. The final concentration is determined by multiplying the absorbance value by the conversion factor and then by the dilution factor used.
Using Absorption Ratios to Determine Sample Purity
While the A260 reading gives the total nucleic acid concentration, a sample’s purity is equally important for successful downstream applications. Spectrophotometry allows scientists to assess purity by measuring absorption at other wavelengths and calculating specific ratios.
A260/A280 Ratio
The A260/A280 ratio is the standard purity check, comparing the nucleic acid peak to the absorption peak of common contaminants, specifically proteins. Proteins absorb UV light most strongly at 280 nm because of the aromatic amino acids they contain, primarily tryptophan and tyrosine. For a pure double-stranded DNA sample, the expected A260/A280 ratio is between 1.8 and 2.0. A ratio significantly lower than 1.8 indicates protein contamination, as the high protein content increases the 280 nm reading.
A260/A230 Ratio
The second purity metric is the A260/A230 ratio, used to detect contamination by various organic compounds and salts carried over from the DNA extraction process. These contaminants, which include phenol, guanidine salts, and certain carbohydrates, absorb light strongly near the 230 nm wavelength. For a clean DNA sample, the A260/A230 ratio is typically expected to be higher than the A260/A280 ratio, falling within the range of 2.0 to 2.2 or sometimes higher. A low A260/A230 ratio suggests a high level of contamination by these chemical residues, which can interfere with enzymatic reactions like PCR or sequencing. Monitoring both ratios provides a comprehensive quality check, ensuring the DNA is sufficiently clean for reliable experimental use.