DNA absorbance intensity measurement


DNA extraction

From Wikipedia, the free encyclopedia
For the various methods, see Nucleic acid methods.

DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and two optional steps in a DNA extraction:

  1. Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by chemical and physical methods-blending, grinding or sonicating the sample.
  2. Removing membrane lipids by adding a detergent or surfactants.
  3. Removing proteins by adding a protease (optional but almost always done).
  4. Removing RNA by adding an RNase (often done).
  5. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. See ethanol precipitation.

Refinements of the technique include adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNase from degrading the DNA.

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water.

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[edit]Special Types of DNA Extractions

A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and any viral episomes present in the cell.

[edit]Detecting DNA

A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

Using the Southern blot technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis.

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