Genomic DNA: Purification

Abstract

Genomic deoxyribonucleic acid (DNA) can be prepared from any source by three steps: cell lysis, deproteinisation, and recovery of DNA. The basic protocol needs to be adapted to the demands of the application, the number of samples to be processed, the requested yield, purity and molecular weight of the DNA, and the amount and history of the source. Traditional protocols based on the lysis with sodium dodecyl sulfate/proteinase K, extraction with phenol/chloroform/isoamyl alcohol, purification with ethanol and storage in Tris/ethylenediamine tetraacetic acid buffer are recommended for the purification of long‐term stable high‐molecular weight genomic DNA from freshly obtained specimens. Intact chromosomal DNA is recovered from agarose‐embedded cells. Automated extraction methods are preferred for forensic applications and high‐throughput processing for biobanking. The technically challenging recovery of ancient DNA needs to be optimised on a case‐to‐case basis, because ancient DNA is damaged and chemically modified and contains large amounts of polymerase chain reaction inhibitors.

Key Concepts:

  • The analysis of complex genomes requires the preparation of pure genomic deoxyribonucleic acid (DNA) of high molecular weight.

  • Genomic DNA can be prepared from any source by three steps: cell lysis, deproteinisation and recovery of DNA.

  • Intact chromosomal megabase‐sized DNA is purified in solid agarose to protect the DNA during preparation.

  • DNA extraction for biobanking relies on automated systems and robotics.

  • Automated systems have been increasingly utilised for DNA extraction by forensic laboratories to handle the growing numbers of forensic casework samples while minimising the risk of human errors and assuring high reproducibility.

  • The recovery of ancient DNA from palaeontological specimens is a demanding task, because ancient DNA is damaged and chemically modified and contains large amounts of PCR inhibitors that interfere with DNA amplification that are copurified with the DNA.

Keywords: agarose‐embedded DNA ; ancient DNA ; biobanking; forensic medicine; palaeontology; high molecular weight DNA

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Further Reading

Carpi FM , Di Pietro F , Vincenzetti S , Mignini F and Napolioni V (2011) Human DNA extraction methods: patents and applications. Recent Patents on DNA and Gene Sequences 5: 1–7.

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How to Cite close
Tümmler, Burkhard, and Stanke, Frauke(Sep 2013) Genomic DNA: Purification. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005330.pub2]