Nucleic acid quantitation - Wikipedia
UV spectra of viruses are complicated by overlapping protein and RNA absorbance ranges from bacteriophages to retroviruses; they package RNA and DNA. a λ−4 relationship between light scattering and wavelength. The ratio of absorbance at nm and nm is used to assess the purity of DNA and RNA. A ratio of ~ is “pure” for DNA; a ratio of ~ is generally accepted as “pure” for RNA. sorbs in the UV both at nm and ~ nm ( Figure 3). In molecular biology, quantitation of nucleic acids is commonly performed to determine the In the case of DNA and RNA, a sample is exposed to ultraviolet light at a wavelength of nanometres (nm) and a The following absorbance units to nucleic acid concentration conversion factors are used to convert OD to.
The UV Absorbance Spectra of Nucleic Acids and Proteins Most biological molecules do not intrinsically absorb light in the visible range, but they do absorb ultraviolet light. It is possible to quantify the amount of DNA in a sample by looking at its absorbance at a wavelength of nm or nm in the UV region. The UV method described in this exercise is not highly accurate but it is very widely used since it is easy, quick, and little DNA is required.
Proteins have two absorbance peaks in the UV region, one between nm, where peptide bonds absorb, and another at about nm due to light absorption by aromatic amino acids tyrosine, tryptophan and phenylalanine. Certain of the subunits of nucleic acids purines have an absorbance maximum slightly below nm while others pyrimidines have a maximum slightly above nm.
Therefore, although it is common to say that the absorbance peak of nucleic acids is nm, in reality, the absorbance maxima of different fragments of DNA vary somewhat depending on their subunit composition. Observe that although proteins have little absorbance at nm, both proteins and nucleic acids absorb light at nm. Therefore, if nucleic acids and proteins are mixed in the same sample, their spectra interfere overlap with one another.
The absorbance spectrum for a mixture of DNA and protein. Distinct DNA and protein peaks cannot be resolved.DNA 260280 Absorbance Reading
Purines have a higher molar absorptivity than pyrimidines. Therefore, the absorbance maximum and absorptivity of a segment of DNA depends on its base composition. Proteins have little absorbance at this wavelength.
It is possible to determine the concentration of nucleic acids or proteins based on their absorbance at a wavelength of nm or nm respectively.
Understanding and measuring variations in DNA sample quality
A calibration curve using standards of known concentration can be constructed. For accurate results, the standard curve should be prepared using the protein of interest or DNA that is similar to that in the sample being measured. Depending on the protein, UV analysis of proteins at nm has a linear range from about 0. The average absorptivity constants for proteins and nucleic acids lead to the following relationships: Values for proteins vary.
A Laboratory Manual", by E. Academic Press, New York. Estimation of the Purity of a Nucleic Acid Preparation It is possible to use UV spectrophotometry to estimate the purity of a solution of nucleic acids.
This method involves measuring the absorbance of the solution at two wavelengths, usually nm and nm, and calculating the ratio of the two absorbances: A ratio less than 1.
Table 2 below summarizes the various UV methods described in this exercise. To compensate for slight turbidity, a background correction can be used.
Why does denatured DNA absorb more ultraviolet light than double-stranded DNA?
Proteins and nucleic acids do not absorb at nm. Therefore, if a sample absorbs at nm, the absorbance is due to turbidity. The absorbance at can be subtracted from the readings at nm and nm: Effect of Acid Under acidic conditions or at extremely high temperatures nucleic acids are completely hydrolysed. At a lower pH, for example pH 3—4 the most easily hydrolysed bonds are broken. These are the glycosylic bonds which attach purine bases to the ribose ring.
Effect of alkali If the pH increases above pH 7—8 the tautomeric state of the bases is affected. This is when some of the hydrogen atoms associated within a base change their location producing a tautomer. These tautomers can form non-standard base pairs that fit into the double helix and can cause the introduction of mutations during DNA replication.
They also change the specific hydrogen bonding between base pairs and destabilise the structure of dsDNA. This change in structure results in a change of absorption at nm as shown in Table 2.
Effects of solvents Absorption of nucleic acids depends on the solvent used to dissolve them. Reports in the literature have compared solubilising DNA in water with a low salt buffer e.
Understanding and measuring variations in DNA sample quality | Articles | Oxford Gene Technology
This is most likely due to differences in the pH of the water caused by the solvation of CO2 from air. As DNA can be difficult to solubilise in water thorough mixing may be needed. Quantification of DNA The absorbance at nm is used to calculate the concentration of nucleic acids. The absorbance value is also dependent on the amount of secondary structure in the DNA due to hypochromicity. To ensure the concentration reading is accurate, the absorbance reading should be within the linear range of the spectrophotometer.
The Lambert-Beer law relates the absorption of light to the properties of the material through which the light is travelling. The law states that there is a logarithmic dependence between the transmission of light through a substance and the product of the absorption coefficient of the substance and the distance the light travels through the material i.
UV Spectrophotometry of DNA, RNA, and Proteins
The law tends to break down at very high concentrations, especially if the material is highly scattering. So for concentrated solutions the absorbance value and therefore the concentration can be inaccurate. It is often useful to prepare and measure a series of dilutions to check not only the concentration but the accuracy of the dilutions as well. Inaccurate dilutions can occur if the DNA is not homogeneously re-suspended.
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