Methods based on absorbance

UV Spectrophotometry using absorbance at 260 nm

Absorbance has been the method of choice for routine quantification of DNA and RNA since decades. It is simple and convenient to use as no further sample treatment (other than DNA extraction) or reaction with other substances is required. However, it is not very specific (it measures all nucleic acids as a whole) and sensitive to contaminants, so it demands very pure DNA to be accurate. Learn more about DNA purity, how to measure it and how it affects quantification.

Absorbance of DNA samples at 260 nm is currently most often measured using a microvolume spectrophotometer, but it is also possible to use a cuvette spectrophotometer or a microplate reader.

In this method, the concentration of a substance is calculated according to the Beer-Lambert law based on its absorbance. The following formula can be obtained from the original formulation of the law:

The formula of the Beer-Lamber law

Where A = absorbance at a given wavelength, ε = extinction coefficient, b = pathlength of the spectrophotometer, c = concentration of the sample.

Hence, for a pathlength of 1 cm, the concentration is equal to the absorbance at 260 nm (the absorption peak of nucleic acids), divided by the extinction coefficient.

Formula to calculate DNA concentration

dsDNA has an extinction coefficient of 0.02 (µg/mL)-1 cm-1, hence:

Recommended formula to calculate the concentration of DNA using absorbance

The same formula can be used with the respective extinction coefficients for ssDNA (absorbance x 37 µg/mL) and ssRNA (absorbance x 40 µg/mL). However, it is important to note that the formula is only valid for large nucleic acid molecules with a similar proportion of all nucleotides, such as genomic DNA, plasmids, etc. For oligonucleotides and other short nucleic acid molecules such as miRNAs, the extinction coefficient has to be calculated from the sequence of the oligonucleotide.

The diphenylamine method (Dische's Test)

Another method of quantifying DNA by absorbance is the diphenylamine method, which is based on the reaction of diphenylamine with deoxyribose sugar to form a blue complex. This method is time-consuming, has a low sensitivity and is therefore no longer used in most laboratories. However, the measurement is made in the visible range and can be performed at 595 nm with a standard ELISA reader if no other instruments are available.