DNA extraction 1053500 215422675 2008-05-28T03:10:48Z 68.33.211.247 /* Detecting DNA */ '''DNA extraction''' is a routine procedure to collect [[DNA]] for subsequent [[molecular biology|molecular]] or [[forensics|forensic]] analysis. There are three basic steps in a DNA extraction: # Breaking the cells open to expose the DNA within, such as by grinding or [[sonication|sonicating]] the sample. # Removing membrane lipids by adding a [[detergent]]. # Precipitating the DNA with an alcohol &mdash; usually [[ethanol]] or [[isopropanol]]. Since DNA is insoluble in these alcohols, it will aggregate together, giving a ''pellet'' on centrifugation. This step also removes alcohol-soluble salt. Refinements of the technique include adding a [[chelating agent]] to sequester divalent cations such as [[magnesium|Mg<sup>2+</sup>]] and [[calcium|Ca<sup>2+</sup>]]. This stops [[dnase]] enzymes from degrading the DNA. Cellular and [[histone]] proteins bound to the DNA can be removed prior to its precipitation either by adding a [[protease]] or having prior to precipitation, precipitating with [[sodium acetate|sodium]] or [[ammonium acetate]], or [[phenol-chloroform extraction|extracting with a phenol-chloroform]] mixture. If desired, the DNA can be resolubilized in a slightly alkaline buffer. {{for|the various methods|Nucleic acid methods}} ==Detecting DNA== {{main|Quantification of nucleic acids}} A diphenylamine (DPA) indicators will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95<sup>o</sup>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 [[Quantification of nucleic acids|260 nm and 280nm]] is used as a measure of DNA purity. DNA absorbs [[UV]] light at 260 and 280 nm, and aromatic proteins absorbs 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 electrophoresis|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 and identification. ==See also== *[[Polymerase chain reaction]] *[[DNA fingerprinting]] *[[Forensic DNA analysis]] *[[Ethanol precipitation]] ==External links== *[http://gslc.genetics.utah.edu/units/activities/extraction/ How to extract DNA from anything living] {{Molecular Biology}} [[Category:Molecular biology]] [[Category:Laboratory techniques]] [[Category:DNA]] [[Category:Polymerase chain reaction]] [[fr:extraction d'ADN]] [[id:Isolasi DNA]] [[lt:DNR išskyrimas]] [[zh:DNA提取]]