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Hydrodynamic shear breakage of DNA

1972; Wiley; Volume: 11; Issue: 12 Linguagem: Inglês

10.1002/bip.1972.360111217

1097-0282

Ray D. Bowman, Norman Davidson,

Protein purification and stability

Abstract The rate of breakage of duplex DNA molecules by laminar flow through a capillary has been studied. For λ b2b5c DNA (molecular wt., M = 25 × 10 6 ) the point at which breakage occurs is normally distributed around the center of the molecule with a standard deviation of 12.5% of the molecular length. At constant shear stress or shear rate, the breakage rate is independent of ionic strength. Thus, shear induced local denaturation is not a rate limiting, preliminary step in breakage. In experiments at constant temperature with varying solvent viscosity (controlled by added sucrose) the breakage rate is a function of shear rate, not of shear stress. The rate of opening of hydrogenbonded circles into linear molecules by hydrodynamic shear is also shown to be a function of shear rate and not of shear stress. The breakage rate at constant shear rate is not greatly dependent on temperature. The shear rate required to achieve breakage is inversely proportional to M 1,2 . The breakage rate constant, k varies as a very high power of the shear rate; at 25°C, d In k/d In Gm ∼ 15; at 10°C, d In k/d In Gm ∼ 26, where Gm is the maximum shear rate at the capillary wall. The unexpected result that breakage rate is mainly dependent on shear rate, not shear stress, supports a model in which the DNA molecule is distorted with a driving force which depends on the hydrodynamic shear stress, η G , but the rate limiting step is segment diffusion into a highly extended configuration. The characteristic time to achieve this configuration is proportional to solvent viscosity, η, hence the breakage rate is dependent on η G/ η or G , the shear rate.