Home   Chapter Home   Jobs   Conferences   Fellowships   Books



Advertisement

Molecular Pathology

DNA purification

Basic Protocol


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.

General
=========================================================================

● 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


This information is intended for physicians and related personnel, who understand that medical information is often imperfect, and must be interpreted in the context of a patient's clinical data using reasonable medical judgment. This website should not be used as a substitute for the advice of a licensed physician.

All information on this website is protected by copyright of PathologyOutlines.com, Inc. Information from third parties may also be protected by copyright. Please contact us at copyrightPathOut@gmail.com with any questions (click here for other contact information).