Quantification of nucleic acids is commonly used in molecular biology to determine the concentrations of DNA or RNA present in a mixture, as subsequent reactions or protocols using a nucleic acid sample often require particular amounts for optimum performance. Molecular biology is the study of Biology at a molecular level Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units A nucleic acid is a Macromolecule composed of chains of monomeric Nucleotides In Biochemistry these Molecules carry Genetic information There exist several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.
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Spectrophotometric quantification
Because DNA and RNA absorb ultraviolet light, with an absorption peak at 260nm wavelength, spectrophotometers are commonly used to determine the concentration of DNA in a solution. In Physics, absorption of electromagnetic radiation is the process by which the Energy of a Photon is taken up by matter typically the electrons of an Ultraviolet ( UV) light is Electromagnetic radiation with a Wavelength shorter than that of Visible light, but longer than X-rays In Physics, spectrophotometry is the quantitative study of electromagnetic spectra. Inside 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. Opacity is the measure of impenetrability to electromagnetic or other kinds of radiation especially visible Light. 020 (μg/ml)-1 cm-1, for single-stranded DNA and RNA it is 0. 027 (μg/ml)-1 cm-1 and for short single-stranded oligonucleotides it is dependent on the length and base composition. Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. In Optics, density is a unitless measure of the Transmittance of an optical element for a given length at a given Wavelength λ: This method of calculation is valid for up to an OD of at least 2. [1] A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model. Opacity is the measure of impenetrability to electromagnetic or other kinds of radiation especially visible Light. [2]
Sample purity
It is common for nucleic acid samples to be contaminated with other molecules (eg, protein, phenol, and other organic compounds). Because these molecules have their own characteristic absorption spectra, the absorption at other wavelengths is often compared to 260nm absorption in order to assess sample purity. In addition, some contaminants (notably phenol) can significantly contribute to an error in concentration estimation as they also absorb strongly at 260nm. Phenol, is a toxic colourless Crystalline Solid with a sweet tarry odor commonly referred to as a "hospital smell"
Protein contamination and the 260:280 ratio
The ratio of absorptions at 260nm vs 280nm is commonly used to assess the purity of DNA with respect to protein contamination, since protein (in particular, the aromatic amino acids) tends to absorb at 280nm. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl The method dates back to 1942, when Warburg and Christian showed that the ratio is a good indicator of nucleic acid contamination in protein preparations. [3] Unfortunately, the reverse is not true -- it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio. [4]
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 of 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 phenol, which is commonly used in nucleic acid purification, can significantly throw off quantification estimates. Phenol, is a toxic colourless Crystalline Solid with a sweet tarry odor commonly referred to as a "hospital smell" Phenol absorbs with a peak at 270nm and a 260:280 ratio of 2. Nucleic acid preparitions uncontaminated by phenol should have a 260:270 ratio of around 1. 2. [1] 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. Phenol, is a toxic colourless Crystalline Solid with a sweet tarry odor commonly referred to as a "hospital smell" The cyanate Ion is an Anion consisting of one Oxygen Atom, one Carbon atom and one Nitrogen atom − in that For a pure nucleic acid sample, the 260:230 ratio should be around 2.
- 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 fluorescence intensity of dyes that bind to nucleic acids and selectively fluoresce when bound (eg. Fluorescence is a Luminescence that is mostly found as an Ethidium bromide). Ethidium bromide (sometimes abbreviated as "EtBr", the abbreviation also confusingly used for Bromoethane) is an intercalating agent 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. Agarose Gel electrophoresis is a method used in Biochemistry and Molecular biology to separate DNA, or RNA molecules by size Plastic wrap is a thin plastic film typically used for sealing Food items in containers to keep them fresh 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. A Polyacrylamide Gel is a separation matrix used in electrophoresis of Biomolecules, such as Proteins or DNA fragments As with the spot test, concentration is estimated through comparison of fluorescent intensity with the known samples. [1]
References
- ^ a b c Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press.
- ^ 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:. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.
- ^ Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press. (Sambrook and Russell cites the original paper: Warburg, O. and Christian W. (1942). "Isolierung und Kristallisation des Gärungsferments Enolase". Biochem. Z. 310: 384–421. )
- ^ Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press. (Sambrook and Russell cites the paper: Glasel J. (1995). "Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios". BioTechniques 18: 62–63. )
External links
- Hillary Luebbehusen, The significance of 260/230 Ratio in Determining Nucleic Acid Purity (pdf document)
See Also
Nucleic acid methods are the techniques used to study Nucleic acids ( DNA and RNA Phenol-chloroform extraction is a Liquid-liquid extraction technique in Biochemistry. Column-based Nucleic acid Purification is a Solid phase extraction method to quickly purify Nucleic acids.Dapyx Software network: MP3 Explorer | Ebook Manager | Zenithic