Analyzing DNA

Spectrophotometric measurement of DNA concentration

DNA concentration can be determined by measuring the absorbance at 260 nm (A260) using a quartz cuvette in a spectrophotometer. For the greatest accuracy, readings should be between 0.1 and 1.0. An absorbance of 1 unit at 260 nm corresponds to 50 µg genomic DNA per mL (A260 =1 for 50 µg/mL; based on a standard 1 cm path length. This relation is valid only for measurements made at neutral pH; therefore, samples should be diluted in a low-salt buffer with neutral pH (e.g., Tris·Cl, pH 7.0). For an example of the calculation used to quantify nucleic acids when using a spectrophotometer, see Spectrophotometric measurement of DNA concentration.

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

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
1 A260 unit Concentration (µg/ml)*
dsDNA 50
 ssDNA 33
Oligonucleotides 20–30

When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, it may be more effective to use agarose gel analysis for quantification (see section Agarose gel).

Tip: If you use more than one cuvette to measure multiple samples, the cuvettes must be matched.

Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration.

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 because of an upward shift in the A260 value.

Effects of solvents on spectrophotometric readings

Absorption of nucleic acids depends on the solvent used to dissolve the nucleic acid (7). A260 values are reproducible when using low-salt buffer, but not when using water. This is most likely due to differences in the pH of the water caused by the solvation of CO2 from air. A2260/A280 ratios measured in water also give rise to a high variability between readings (see figure Effect of solvent on A2260/A280 ratio) and the ratios obtained are typically <1.8, resulting in reduced sensitivity to protein contamination (7). In contrast, A2260/A280 ratios measured in a low-salt buffer with slightly alkaline pH are generally reproducible.

Effect of solvent on A2260/A280 ratio
Effect of RNA contamination on spectrophotometric readings

Depending on the DNA isolation method used, RNA will be co-purified with genomic DNA. RNA may inhibit some downstream applications, but it will not inhibit PCR. Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration. You can sometimes detect RNA contamination using agarose gel analysis with routine ethidium bromide staining, although it’s not possible to quantify the amount effectively. RNA bands will appear faint and smeary and are only detected in amounts ≥25–30 ng (0.5:1 RNA:DNA ratio).

To effectively remove contaminating RNA, treat your samples with RNase A. Either incorporate it into the purification procedure or after the DNA has been purified. Before use, ensure the RNase A solution has been heat-treated to destroy any contaminating DNase activity. Alternatively, use DNase-free RNase purchased from a reliable supplier.

Purity of DNA

The ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate of DNA purity with respect to contaminants that absorb UV light, such as protein. The A260/A280 ratio is influenced considerably by pH. Since water is not buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results in a lowerA260/A280 ratio and reduced sensitivity to protein contamination (7). For accurate A260/A280 values, we recommend measuring absorbance in a slightly alkaline buffer (e.g., 10 mM Tris·Cl, pH 7.5). Be sure to zero the spectrophotometer with the appropriate buffer. 

Pure DNA has an A260/A280 ratio of 1.7–1.9. If you scan the absorbance from 220–320 nm, you can check whether there are contaminants affecting absorbance at 260 nm. Absorbance scans should show a peak at 260 nm and an overall smooth shape.

Fluorometry allows you to specifically and sensitively measure the DNA concentration using a fluorescent dye. Common dyes include Hoechst dyes and PicoGreen.

Hoechst 33258 has little affinity for RNA, allowing accurate quantification of DNA samples contaminated with RNA. It shows increased emission at 458 nm when bound to DNA. DNA standards and samples are mixed with Hoechst 33258 and measured in glass or acrylic cuvettes using a scanning fluorescence spectrophotometer or a dedicated filter fluorometer set at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. Compare the sample measurements to the standards to determine the DNA concentration.

Tip: As Hoechst 33258 preferentially binds AT-rich DNA, use standards with a similar base composition to the sample DNA.

PicoGreen is a highly sensitive measure of dsDNA and can measure as little as 20 pg dsDNA in a 200 µL assay volume. Indeed, DNA concentrations from 500 pg/mL to 500 ng/mL can all be measured using a single dye concentration. The assay is optimized to minimize the fluorescence contributions of RNA and ssDNA, allowing accurate quantification of dsDNA in the presence of equimolar concentrations of ssDNA and RNA with minimal effect on the quantitative results.

Agarose gel analysis enables quick and easy quantification of DNA, especially for small DNA fragments (such as PCR products). As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining. The DNA sample is run on an agarose gel alongside known amounts of DNA of the same or a similar size. The amount of sample DNA loaded can be estimated by comparison of the band intensity with the standards either visually (see figure Agarose gel analysis of plasmid DNA) or using a scanner or imaging system. Be sure to use standards of roughly the same size as the fragment of interest to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity.

More precise agarose gel quantification can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. In most experiments the effective range for comparative densitometric quantification is between 20 and 100 ng.

Tip: The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve.

See DNA analysis using analytical gels, for further information on agarose gel electrophoresis.