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Team:Alberta/References/Publications/The effect of hydrodynamic shear of DNA from T2 and T4 bacteriophages
From 2009.igem.org
M.S. Levy, IJ. Collins, S.S. Yim, J.M. Ward, N. Titchener-Hooker, P. Ayazi Shamlou, and P. Dunnill
Biotechnology and Bioengineering Volume 87 Issue 3, Pages 293 - 302
Abstract: This study was designed to evaluate the effect of
shear on the supercoiled circular (SC) form of plasmid
DNA. The conditions chosen are representative of those
occurring during the processing of plasmid-based genes for
gene therapy and DNA vaccination. Controlled shear was
generated using a capillary rheometer and a rotating disk
shear device. Plasmid DNA was tested in a clari®ed alkaline
lysate solution. This chemical environment is characteristic
of the early stages of plasmid purification. Quantitative
data is reported on shear degradation of three homologous
recombinant plasmids of 13, 20 and 29 kb in size. Shear
sensitivity increased dramatically with plasmid molecular
weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/
HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear
using the capillary rheometer. The shear sensitivity of the
three plasmids was similar to that observed for the same
plasmids in the clarified alkaline lysate. Further experiments
were carried out using the 20 kb plasmid and the
rotating disk shear device. In contrast with the capillary
rheometer data, ultrapure DNA redissolved in TE buffer
was up to eight times more sensitive to shear compared to
plasmid DNA in the clarified alkaline lysate. However, this
enhanced sensitivity decreased when the ionic strength of
the solution was raised by the addition of NaCl to 150 mM.
In addition, shear damage was found to be independent of
plasmid DNA concentration in the range from 0.2 lg/ml to
20 lg/ml. The combination of shear and air-liquid interfaces
caused extensive degradation of the plasmid DNA.
The damage was more evident at low ionic strength and low
DNA concentration. These findings show that the tertiary
structure of plasmid DNA can be severely affected by shear
forces. The extent of damage was found to be critically
dependent on plasmid size and the ionic strength of the
environment. The interaction of shear with air-liquid interfaces
shows the highest potential for damaging SC
plasmid DNA during bioprocesses.
Link: Sciencedirect
