Quantification of nucleic acids
Encyclopedia
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 acid
s 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.
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 (estimation 0.030 (μg/ml)-1 cm-1). Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an OD of at least 2. A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.
, array CGH, qPCR) imply quantitative and qualitative nucleic acid analysis with minimal sample volumes. Specialized NanoPhotometer offer the possibility to determine sample concentrations cuveteless with submicroliter volumes (starting with 0.3 µl). In addition, due to the reduction of the optical pathlength samples are diluted automatically in comparison to standard cuvette measurements. The respective virtual dilution factors are considered by the software of the instrument. Because the measurements are processed with undiluted samples, the reproducibility of the results is very high. And if desired, samples can be retrieved after the measurement for further processing.
solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280nm. 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.
260:280 ratio has high sensitivity for nucleic acid contamination in protein:
260:280 ratio lacks sensitivity for protein contamination in nucleic acids:
This difference is due to the much higher extinction coefficient nucleic acids have at 260nm and 280nm, 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.
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 260nm 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.
If the sample volumes are large enough to use microplates
or cuvette
s, the dye-loaded samples can also be quantified with a fluorescence photometer
.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, quantitation of nucleic acids is commonly performed to determine the average concentrations of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
present in a mixture, as well as their purity. Reactions that use nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s 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
Nucleic acids absorbAbsorption (electromagnetic radiation)
In physics, absorption of electromagnetic radiation is the way by which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed to other forms of energy for example, to heat. The absorption of light during wave propagation is...
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 (estimation 0.030 (μg/ml)-1 cm-1). Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an OD of at least 2. A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model.
Conversion Factors for Nucleic Acids
| Nucleic Acid | Concentration (μg/ml) for 1 A260 unit |
|---|---|
| dsDNA | 50 |
| ssDNA | 37 |
| ssRNA | 40 |
Cuvette nucleic acid quantitation
The optical density of samples measured with 10 mm path length standard cuvettes simply has to be multiplied by the conversion factor to determine the concentration of the sample. Example, a dsDNA sample with an OD of 0.9 corresponds to a sample concentration of 45 µg/ml.Cuvetteless low volume nucleic acid quantitation
Multiple biological applications (e.g. DNA microarray experimentDNA microarray experiment
This is an example of a DNA microarray experiment, detailing a particular case to better explain DNA microarray experiments, while enumerating possible alternatives.# The two samples to be compared are grown/acquired...
, array CGH, qPCR) imply quantitative and qualitative nucleic acid analysis with minimal sample volumes. Specialized NanoPhotometer offer the possibility to determine sample concentrations cuveteless with submicroliter volumes (starting with 0.3 µl). In addition, due to the reduction of the optical pathlength samples are diluted automatically in comparison to standard cuvette measurements. The respective virtual dilution factors are considered by the software of the instrument. Because the measurements are processed with undiluted samples, the reproducibility of the results is very high. And if desired, samples can be retrieved after the measurement for further processing.
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 280nm (A260/280) is used to assess the purity of nucleic acids. For pure DNA, A260/280 is ~1.8 and for pure RNA A260/230 is ~2.Protein contamination and the 260:280 ratio
The ratio of absorptions at 260nm vs 280nm is commonly used to assess DNA contamination of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280nm. 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.
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 |
260:280 ratio lacks sensitivity for protein contamination in nucleic acids:
| % nucleic acid | % protein | 260:280 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 260nm and 280nm, 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
- Contamination by phenolPhenolPhenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
, which is commonly used in nucleic acid purification, can significantly throw off quantification estimates. Phenol absorbs with a peak at 270nm and a A260/280 of 1.2. Nucleic acid preparations uncontaminated by phenol should have a A260/280 of around 2. Contamination by phenol can significantly contribute to overestimation of DNA concentration. - Absorption at 230nm can be caused by contamination by phenolate ion, thiocyanates, and other organic compounds. For a pure RNA sample, the A260/230 should be around 2, and for a pure DNA sample, the A260/230 should be around 1.8.
- Absorption at 330nm and higher indicates particulates contaminating the solution, causing scattering of light in the visible range. The value in a pure nucleic acid sample should be zero.
- Negative values could result if an incorrect solution was used as blank. Alternatively, these values could arise due to fluorescence of a dye in the solution.
Quantification using fluorescent dyes
An alternative way to assess DNA concentration is to use measure the fluorescenceFluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
intensity of dyes that bind to nucleic acids and selectively fluoresce when bound (e.g. Ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
). This method is useful for cases where concentration is too low to accurately assess with spectrophotometry and in cases where contaminants absorbing at 260nm 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
Plastic wrap
Plastic wrap, cling film , cling wrap or food wrap, is a thin plastic film typically used for sealing food items in containers to keep them fresh over a longer period of time...
. 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.
If the sample volumes are large enough to use microplates
Microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
or cuvette
Cuvette
A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...
s, the dye-loaded samples can also be quantified with a fluorescence photometer
Photometer
In its widest sense, a photometer is an instrument for measuring light intensity or optical properties of solutions or surfaces. Photometers are used to measure:*Illuminance*Irradiance*Light absorption*Scattering of light*Reflection of light*Fluorescence...
.
See also
- Nucleic acid methodsNucleic acid methodsNucleic acid methods are the techniques used to study nucleic acids .Purification*Phenol-chloroform extraction*minicolumn purification*RNA extractionQuantification*Abundance in weight: spectroscopic quantification...
- RNA quality control
- Phenol-chloroform extraction
- Column purification
External links
- IDT online tool for predicting nucleotide UV absorption spectrum
- Ambion guide to RNA quantitation
- Hillary Luebbehusen, The significance of 260/230 Ratio in Determining Nucleic Acid Purity (pdf document)
- double stranded, single stranded DNA and RNA quantification by 260nm absorption, Sauer lab at OpenWetWare
- Absorbance to Concentration Web App @ DNA.UTAH.EDU