Molecular markers
DNA purification
Basic protocol

Author: Rodney E. Shackelford, D.O., Ph.D. (see Authors page)

Revised: 29 May 2018, last major update April 2012

Copyright: (c) 2008-2018, PathologyOutlines.com, Inc.

PubMed Search: Molecular DNA purification basic protocol

Table of Contents
Definition / general
Cite this page: Shackelford, R.E. Basic protocol. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurprotocol.html. Accessed September 25th, 2018.
Definition / 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:
    1. 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
    2. Add divalent cation chelators to bind and sequester Mg2+ and Ca2+, which are often required for DNase activities
    3. 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