Nucleic acid quantitation

In molecular biology, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA or RNA present in a mixture, as well as their purity. Reactions that use nucleic acids often require particular amounts and purity for optimum performance. There are several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.

Spectrophotometric analysis

optical density of Ribosome sample. The important wavelength of 260nm and 280nm are labeled.

Nucleic acids absorb ultraviolet light in a specific pattern. In a spectrophotometer, a sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample.

Using the Beer-Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule. At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/ml)−1 cm−1, for single-stranded DNA it is 0.027 (μg/ml)−1 cm−1, for single-stranded RNA it is 0.025 (μg/ml)−1 cm−1 and for short single-stranded oligonucleotides it is dependent on the length and base composition. Thus, an Absorbance (A) of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an A of at least 2.[1] A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.[2]

Sample purity

It is common for nucleic acid samples to be contaminated with other molecules (i.e. proteins, organic compounds, other). The ratio of the absorbance at 260 and 280 nm (A260/280) is used to assess the purity of nucleic acids. For pure DNA, A260/280 is widely considered ~1.8 but has been argued to translate - due to numeric errors in the original Warburg paper - into a mix of 60% protein and 40% DNA.[3] The ratio for pure RNA A260/280 is ~2.

Protein contamination and the 260:280 ratio

The ratio of absorbance at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm.[1][4] The reverse, however, is not true — it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio in a nucleic acid solution.[1][3]

260:280 ratio has high sensitivity for nucleic acid contamination in protein:

% protein% nucleic acid 260:280 ratio
100 0 0.57
95 5 1.06
90 10 1.32
70 30 1.73

260:230 ratio lacks sensitivity for protein contamination in nucleic acids (table shown for RNA, 100% DNA is approximately 1.8):

% nucleic acid% protein 260:230 ratio
100 0 2.00
95 5 1.99
90 10 1.98
70 30 1.94

This difference is due to the much higher extinction coefficient nucleic acids have at 260 nm and 280 nm, compared to that of proteins. Because of this, even for relatively high concentrations of protein, the protein contributes relatively little to the 260 and 280 absorbance. While the protein contamination cannot be reliably assessed with a 260:280 ratio, this also means that it contributes little error to DNA quantity estimation.

Other common contaminants

Quantification using fluorescent dyes

An alternative way to assess DNA concentration is to measure the fluorescence intensity of dyes that bind to nucleic acids and selectively fluoresce when bound (e.g. Ethidium bromide). This method is useful for cases where concentration is too low to accurately assess with spectrophotometry and in cases where contaminants absorbing at 260 nm make accurate quantitation by that method impossible.

There are two main ways to approach this. "Spotting" involves placing a sample directly onto an agarose gel or plastic wrap. The fluorescent dye is either present in the agarose gel, or is added in appropriate concentrations to the samples on the plastic film. A set of samples with known concentrations are spotted alongside the sample. The concentration of the unknown sample is then estimated by comparison with the fluorescence of these known concentrations. Alternatively, one may run the sample through an agarose or polyacrylamide gel, alongside some samples of known concentration. As with the spot test, concentration is estimated through comparison of fluorescent intensity with the known samples.[1]

If the sample volumes are large enough to use microplates or cuvettes, the dye-loaded samples can also be quantified with a fluorescence photometer. Minimum sample volume starts at 0.3 μl [6]

See also

References

  1. 1 2 3 4 5 Sambrook & Russell (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press. ISBN 978-0-87969-577-4.
  2. ↑ Tataurov A.V.; You Y.; Owczarzy R. (2008). "Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids". Biophys. Chem. 133 (1-3): 66–70. doi:10.1016/j.bpc.2007.12.004. PMID 18201813.
  3. 1 2 Glasel J. (1995). "Validity of nucleic acid purities monitored by 260/280 absorbance ratios". BioTechniques 18 (1): 62–63. PMID 7702855.)
  4. ↑ (Sambrook and Russell cites the original paper: Warburg, O. & Christian W. (1942). "Isolierung und Kristallisation des Gärungsferments Enolase". Biochem. Z. 310: 384–421.)
  5. ↑ "The Analysis of DNA or RNA using Its Wavelengths: 230 nm, 260 nm, 280 nm". Bioteachnology.com. 2010-01-13. Retrieved 2010-03-12.
  6. ↑ Nucleic Acid Quantification Accuracy and Reproducibility

External links

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