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DNA: Mechanical Breakage

  1. Michael Andrew Quail

Published Online: 15 NOV 2010

DOI: 10.1002/9780470015902.a0005333.pub2

eLS

eLS

How to Cite

Quail, M. A. 2010. DNA: Mechanical Breakage. eLS. .

Author Information

  1. Wellcome Trust Sanger Institute, Cambridge, UK

Publication History

  1. Published Online: 15 NOV 2010

Abstract

In order to fragment deoxyribonucleic acid (DNA) in a random manner, a variety of methods involving the mechanical breakage of DNA have been employed. These include sonication, needle shear, nebulisation, point-sink shearing and passage through a French pressure cell. The new sequencing technologies utilise smaller fragments than those traditionally generated by mechanical breakage. As a result a number of higher throughput and more powerful technologies for random shearing of DNA have been developed including focused acoustic shearing (Covaris Inc) and high power sonication devices (e.g. Sonicman and Bioruptor). These are able to efficiently fragment DNA down to 100 bp and options for multiplexed sample processing are available.

This article describes each of these approaches in turn giving the theory behind and the utility of each and giving practical tips where appropriate.

Key Concepts:

  • Shearing DNA using mechanical breakage produce random fragments.

  • Shearing DNA by utilising enzymatic action generates bias and produces a set of nonrandom fragments.

  • Several genomic analysis procedures, especially sequencing, require the shearing of DNA to a set of random DNA fragments.

  • Heat generated during physical shearing can cause A–T rich regions of DNA to permanently dissociate.

  • Randomly sheared DNA needs ‘repairing’, typically by incubation with Klenow or T4 polymerase in the presence of dNTPs, in order to make their termini available for ligation.

Keywords:

  • shearing;
  • sonication;
  • nebuliser;
  • random breakage;
  • hydroshear