Quantification of DNA презентация

Quantification of DNA

Two methods are widely used to determine the concentration of DNA in solution. The most simple and accurate is the spectrophotometric method, but it has a relatively low sensitivity. If the total content of nucleic acids is small, the concentration of DNA can be determined from the intensity of their fluorescence in UV light after staining with ethidium bromide Two methods are widely used to determine the concentration of DNA in solution. The most simple and accurate is the spectrophotometric method, but it has a relatively low sensitivity. If the total content of nucleic acids is small, the concentration of DNA can be determined from the intensity of their fluorescence in UV light after staining with ethidium bromide a. Electrophoretic Method For an approximate evaluation of the concentration of DNA, different amounts of marker nucleic acids are applied to the gel. The concentration of DNA in the sample is evaluated by comparing the fluorescence intensity of the sample and standard markers with known concentrations. It is important that the DNA samples are compared with the corresponding markers, since for the same amount of DNA the fluorescence intensity in UV light differs. B. Spot Method The concentration of nucleic acids in a solution can be determined by staining the solution with ethidium bromide by measuring the fluorescence intensity in UV light and comparing it with the fluorescence of markers of known concentration. As with the electrophoretic method, the markers must be nucleic acids of the corresponding type (i.e., DNA)

Nucleic acids The rings of the bases (A, C, G, T, U) are made up of alternating single and double bonds. Such ring structures absorb in the U.V. Each of the four nucleotide bases has a slightly different absorption spectrum, and the spectrum of DNA is the average of them.

It operates on Beer’s law: It operates on Beer’s law: When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, the more concentrated the specimen is, the less light is transmitted through it.

DNA Analysis (After extraction) DNA Analysis (After extraction) Nucleic acid (DNA/RNA) Qualitative analysis Quantitative analysis Size-Quality Purity- Yield Agarose gel electrophoresis Spectrophotometry

Automated Sequencing Dye primer or dye terminator sequencing on capillary instruments. Sequence analysis software provides analyzed sequence in text and electropherogram form. Peak patterns reflect mutations or sequence changes.