DNA: Mechanical Breakage

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

Figure 1.

Ethidium bromide stained agarose gel showing typical results of DNA shearing by various techniques: lanes 1 and 9, DNA molecular weight markers (kb ladder, Life Technologies); lane 2, 100 ng untreated genomic DNA; lane 3, DNA sheared by 10 passes through a 30‐gauge needle; lane 4, DNA sheared by sonication; lane 5, DNA sheared using a Hydroshear set at speed code 3; lane 6, DNA sheared by nebulisation at 55.2 kPa (8 psi); lane 7, unsheared 1.75 kb polymerase chain reaction (PCR) product and lane 8, sonicated 1.75 kb PCR product. The DNA used for this comparison was human placental DNA, Sigma catalogue number D4642.

Figure 2.

Cup‐horn sonication probe (model CL4, Misonix Inc., Farmingdale, NY). In the cup‐horn device, a wide, flat, stainless‐steel probe is inverted and has a Perspex chamber built around it in which ice‐cold water can be placed. The DNA sample, typically in a volume of approximately 50–100 μL and contained within a plastic microcentrifuge tube, is held in a clamp such that it is suspended close to the surface of the sonic probe. The probe is then turned on for a set period of time at the predetermined optimal intensity to fragment the DNA.

Figure 3.

Nebuliser unit from IPI Medical Products Inc., Chicago, IL. The unit has a large cone‐shaped central chamber with a narrow central inlet port and a wide outlet port (to which a mouthpiece is attached when used to dispense pharamaceutical products).

Figure 4.

Diagram depicting the process of adaptive acoustic shearing. A cone shaped transducer focuses intense acoustic energy on a sample, contained within a small glass tube, inducing cavitation and shearing the DNA. Reproduced with permission from Covaris Inc.

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References

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Further Reading

Hengen P (1997) Shearing DNA for genomic library construction. Trends in Biochemical Sciences 22: 273–274.

Quail MA, Kozarewa I, Smith F et al. (2008) A large genome center's improvements to the Illumina sequencing system. Nature Methods 5: 1005–1010.

Wilson RK and Mardis ER (1997) Shotgun sequencing. In: Birren B, Green ED, Klapholz S, Myers RM and Roskams J (eds) Genome Analysis: A Laboratory Manual (Volume 1 Analyzing DNA), pp. 398–456. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Web Links

Covaris. Products: S, L and E series AFA shearing platforms‐ Details of products, applications, publications and videos. http://www.covarisinc.com/index.html

Diagenode. Products: Bioruptor–Diagram and recommended protocol for shearing prior to next gen sequencing http://www.diagenode.com/en/topics/sonication/sonication.php

GeneMachines. Products: HydroShear–a description and video of the apparatus http://www.genemachines.com/HydroShear.html

Matrical. Products: Sonicman‐ Product details and application note for DNA shearing http://www.matrical.com/SonicMan_High_Throughput_Sonication.php

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How to Cite close
Quail, Michael Andrew(Nov 2010) DNA: Mechanical Breakage. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005333.pub2]