Analysing DNA

Quantification of DNA

Reliable measurement of DNA concentration is important for many applications in molecular biology. Spectrophotometry and fluorometry are commonly used to measure both genomic and plasmid DNA concentration. Spectrophotometry can be used to measure microgram quantities of pure DNA samples (i.e., DNA that is not contaminated by proteins, phenol, agarose, or RNA). Fluorometry is more sensitive, allowing measurement of nanogram quantities of DNA, and furthermore, the use of Hoechst 33258 dye allows specific analysis of DNA.

DNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer using a quartz cuvette. For 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). An example of the calculation involved in nucleic acid quantification when using a spectrophotometer (see Spectrophotometric measurement of DNA concentration).

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

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

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

Tip: 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. A260/A280 ratios measured in water also give rise to a high variability between readings (see figure Effect of solvent on A260/A280 ratio) and the ratios obtained are typically <1.8, resulting in reduced sensitivity to protein contamination (7). In contrast, A260/A280 ratios measured in a low-salt buffer with slightly alkaline pH are generally reproducible.

Effect of solvent on A260/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. RNA contamination can sometimes be detected by agarose gel analysis with routine ethidium bromide staining, although not quantified effectively. RNA bands appear faint and smeary and are only detected in amounts ≥25–30 ng (0.5:1 RNA:DNA ratio).

Treatment with RNase A will remove contaminating RNA; this can either be incorporated into the purification procedure or performed after the DNA has been purified. Prior to use, ensure that the RNase A solution has been heat-treated to destroy any contaminating DNase activity. Alternatively, use DNase-free RNase purchased from a reliable supplier.

RNA contamination of plasmid DNA can be a concern depending on the method used for plasmid preparation. Methods using alkaline lysis with phenol extraction cannot separate RNA from plasmid DNA, leading to high levels of RNA contamination. Advanced anion-exchange technology allows isolation of high-molecular-weight genomic DNA that is free of RNA.

 

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 lower A260/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. Scanning the absorbance from 220–320 nm will show 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 specific and sensitive measurement of DNA concentration by use of a fluorescent dye; with common dyes including Hoechst dyes and PicoGreen.

Hoechst 33258 has little affinity for RNA, allowing accurate quantification of DNA samples that are 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. The sample measurements are then compared to the standards to determine 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 measures using a single dye concentration. The assay is optimized to minimize the fluorescence contributions of RNA and ssDNA, such that dsDNA can be accurately quantified 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.
Agarose gel analysis of plasmid DNA

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.

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