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. 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 0.020 (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. 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.
260:280 ratio has high sensitivity for nucleic acid contamination in protein:
|% protein||% nucleic acid||260:280 ratio|
260:280 ratio lacks sensitivity for protein contamination in nucleic acids:
|% nucleic acid||% protein||260:280 ratio|
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.
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. 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.
WIPO ASSIGNS PATENT TO QIAGEN FOR "METHOD OF NORMALIZED QUANTIFICATION OF NUCLEIC ACIDS USING ANCHOR OLIGONUCLEOTIDES AND ADAPTER OLIGONUCLEOTIDES" (GERMAN INVENTORS)
Feb 08, 2011; GENEVA, Feb. 8 -- Publication No. WO/2011/012330 was published on Feb. 03. Title of the invention: "METHOD OF NORMALIZED...
Patent Application Titled "Method for the Efficiency-Corrected Real-Time Quantification of Nucleic Acids" under Review
Nov 15, 2012; By a News Reporter-Staff News Editor at Politics & Government Week -- According to news reporting originating from Washington,...
WIPO ASSIGNS PATENT TO QIAGEN FOR "LIGATION-BASED METHOD OF NORMALIZED QUANTIFICATION OF NUCLEIC ACIDS" (GERMAN INVENTORS)
Feb 05, 2011; GENEVA, Feb. 5 -- Publication No. WO/2011/012328 was published on Feb. 03. Title of the invention: "LIGATION-BASED METHOD OF...
Wipo Publishes Patent of Korea Research Institute of Standards and Science, Yang in Chul, Kang Min Jung, Yu Han Nah, Park Sang Ryoul, Kim Sook Kyoung for "Method and Kit for the Quantification of Nucleic Acids" (South Korean Inventors)
Aug 09, 2012; GENEVA, Aug. 9 -- Publication No. WO/2012/070788 was published on May 31. Title of the invention: "METHOD AND KIT FOR THE...