Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2012, PathologyOutlines.com, Inc.
● Initial nucleic acid isolation requires cells lysis or solubilization of nucleic acids in sample
● Nucleic acids are most commonly isolated from whole blood, plasma, white cells, tissue (often formalin-fixed or quick-frozen), feces, urine
Most protocols follow these basic steps:
1. Break/disperse sample into solution; often involves cell lysis to expose the nucleic acids
2. Remove lipid/membrane with various detergents or less commonly alcohols; most samples containing cells will require the separation of cell membranes from nucleic acids
3. Remove proteins by digestion with Protinase K or other protease K
4. To obtain pure DNA, (a) incubate sample with RNase (to digest/remove RNA); RNA can also be removed by increasing alkalinity, causing internal phosphoester transfer reactions, leading to RNA strand cleavage; (b) add divalent cation chelators to bind and sequester Mg2+ and Ca2+, which are often required for DNase activities; (c) additional steps depend on sample type; for example, resins in plants samples can make nucleic acid isolation complex and difficult
5. Are many specific different nucleic acid isolation protocols; often two or more are combined for better nucleic acid purification, such as organic extraction followed by cesium chloride gradient centrifugation
End of Molecular Pathology > DNA Purification > Basic Protocol
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