Analysing DNA

DNA spectrophotometer for measuring purity and concentration

How to determine DNA purity and concentration

The concentration and purity of DNA can be determined using a variety of methods. While the most common approach is to measure the absorbance at 260 nm (A₂₆₀) in a standard DNA spectrophotometer or microvolume instrument, alternative DNA quantification methods may offer higher sensitivity or specificity depending on the application.

DNA quantification by spectrophotometer measures the absorbance at 260 nm (A₂₆₀) in a standard DNA spectrophotometer or microvolume instrument. Determination of the purity and concentration is based on the principle that nucleic acids, including DNA, absorb ultraviolet (UV) light maximally at 260 nm due to their aromatic bases. This absorption follows the Beer-Lambert law, where the absorbance at 260 nm is directly proportional to the DNA concentration.

The ratio of the readings at 260 nm and 280 nm (A₂₆₀/A₂₈₀) provides an estimate of DNA purity. A low ratio indicates the presence of contaminants that absorb UV light, such as protein.

Pure DNA has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0 in 10 mM Tris·Cl, pH 8.5.

Note: The absorbance measurements cannot discriminate between DNA and RNA, and RNA contamination can lead to overestimation of the DNA concentration. However, the A₂₆₀/A₂₈₀ ratios for DNA and RNA are different. Pure RNA gives a reading ~2.0 and pure DNA a reading ~1.8. For example, an A₂₆₀/A₂₈₀ ratio of 1.95 can suggest RNA contamination.

Note: Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, due to an upward shift in the A₂₆₀ value.

Concentration (µg/mL) = (A₂₆₀ reading – A₃₂₀ reading) × dilution factor × 50 µg/mL

The total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (mL)

Protocol to determine DNA purity and concentration

Blank the DNA spectrophotometer with the diluent the sample is in.
Tip: For accurate A₂₆₀/A₂₈₀ values, measure the absorbance in slightly alkaline buffer (e.g., 10 mM Tris·Cl, pH 7.5).
Measure the absorbance of your sample at 260 nm and 280 nm.
Tip: To ensure reliability, readings should be between 0.1 and 1.0.
Take a background reading at 320 nm (A₃₂₀) to correct for turbidity or cloudiness in the sample.
Calculate the DNA purity using the A₂₆₀/A₂₈₀ ratio. A ratio of ~1.8 is generally considered pure for DNA.
Calculate the DNA concentration using the formula:
Concentration (µg/mL) = (A₂₆₀ reading – A₃₂₀ reading) × dilution factor × 50 µg/mL.

Strong absorbance at A₂₈₀ resulting in a low A₂₆₀/A₂₈₀ ratio indicates the presence of contaminants, such as proteins.
Strong absorbance at 270 nm and 275 nm may indicate the presence of contaminating phenol.
Absorbance at 325 nm suggests contamination by particulates in the solution or dirty cuvettes.

Spectrophotometric conversions from absorbance at 260 nm

[A] A highly pure gDNA sample shows no significant contamination as evidenced by the spectrum. Only the blue spectrum for DNA is present, while those for impurities (orange), background (gray) and residue (yellow) are not.

[B] An RNA sample was spiked with gDNA. The RNA is indicated by the blue absorbance line, and the spiked DNA is represented by the orange absorbance line.

[C] An RNA sample was spiked with phenol. The RNA is indicated by the blue absorbance line, and the spiked phenol is represented by the orange absorbance line.

[D] A gDNA sample was spiked with hemoglobin. The gDNA is indicated by the blue absorbance line, and the spiked hemoglobin is represented by the gray absorbance line. The black absorbance curve indicates the total measured, background-corrected spectrum of the sample; the sum of DNA and RNA is shown as a pink absorbance curve (only shown if no differentiation between DNA and RNA can be performed); the blue absorbance line indicates the target molecule (DNA or RNA; app-dependent); the orange spectrum indicates impurities (DNA or RNA, salts, phenol; app-dependent); the gray absorbance curve shows the measured background of the sample (turbidity, beads, hemoglobin, chlorophyll; app-dependent); the yellow line indicates the unknown part of a sample that cannot be assigned to the above mentioned spectral parts.

Standard DNA spectrophotometer versus microvolume spectrophotometer

Microvolume DNA spectrophotometers are attractive due to the low sample volume (1 µl) and convenience, as no cuvettes are required. These enable rapid, accurate concentration and purity measurements of DNA, RNA and protein.

In contrast, a standard DNA spectrophotometer requires a larger sample volume and uses a quartz cuvette with a 1 cm pathlength. It is still a common method for measuring absorbance at 260 nm, particularly if the samples require the established 1 cm pathlength for concentration calculations.

Advantages and disadvantages of DNA spectrophotometry

Spectrophotometric analysis of DNA is a simple method for determining the purity and concentration of DNA – but it has some limitations. It is not possible to distinguish DNA from other nucleic acids, such as RNA. While it provides information about DNA purity and concentration, a highly pure DNA sample is necessary for reliable results.

DNA spectrophotometer versus fluorometer

DNA quantification by spectrophotometer can be used to measure microgram quantities of pure DNA samples (i.e., DNA that is not contaminated by proteins, phenol, agarose, or RNA). In comparison, quantification by fluorometer is more sensitive than spectrophotometric analysis of DNA, allowing measurement of nanogram quantities of DNA. Furthermore, use of the Hoechst 33258 dye in fluorometry allows specific analysis of DNA.