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. To date, there are two main approaches used by scientists to quantitate, or establish the concentration, of nucleic acids (such as DNA or RNA) in a solution. These are spectrophotometric quantification and UV fluorescence tagging in presence of a DNA dye.* High 260/280 purity ratios are not normally indicative of any issues. 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. To date, there are two main approaches used by scientists to quantitate, or establish the concentration, of nucleic acids (such as DNA or RNA) in a solution. These are spectrophotometric quantification and UV fluorescence tagging in presence of a DNA dye. One of the more commonly used practices to quantitate DNA or RNA is the use of spectrophotometric analysis using a spectrophotometer. A spectrophotometer is able to determine the average concentrations of the nucleic acids DNA or RNA present in a mixture, as well as their purity. Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet light in a specific pattern. In the case of DNA and RNA, a sample is exposed to ultraviolet light at a wavelength of 260 nanometres (nm) and a photo-detector measures the light that passes through the sample. Some of the ultraviolet light will pass through and some will be absorbed by the DNA / RNA. The more light absorbed by the sample, the higher the nucleic acid concentration in the sample. The resulting effect is that less light will strike the photodetector and this will produce a higher optical density (OD) 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. A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model. The optical density is generated from equation: In practical terms, a sample that contains no DNA or RNA should not absorb any of the ultraviolet light and therefore produce an OD of 0