Determination of DNA concentration and purity

If the concentration of DNA in the sample is sufficient, it can be measured by direct photometry in the UV region. Due to the large number of purine residues, pure DNA has an absorption maximum at 260 nm, with an absorbance of approximately 1 for a DNA solution with a concentration of 50 μg·ml-1.

At 260 nm, however, proteins also absorb, whose spectrum has a broad peak with a maximum at 280 nm due to tyrosine groups. In practice, DNA purity is often estimated by the absorbance ratio at 260 and 280 nm. Pure DNA has an A260/A280 of approximately 1.8 (the higher the ratio, the purer the DNA).

When working with diluted DNA solutions, direct photometry is not sensitive enough. In that case, intercalation fluorescent dyes are most often used to determine its concentration. They are substances that contain several condensed aromatic nuclei, so they have a planar structure. Thanks to it, they can "wedge", intercalate between the strands of DNA double helices, while increasing their fluorescence. The prototype of intercalation dyes is ethidium bromide (3,5-diamino-5-ethyl-6-phenylphenanthridium bromide).