DNA extraction
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2008-05-28T03:10:48Z
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/* 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 — 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]]
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[[id:Isolasi DNA]]
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[[zh:DNA提取]]