Shear breakage of DNA.
Determinations were made of the mean length of fragments produced after shearing long (greater than 100 kb) native Hela DNA in a VirTis homogenizer. (VirTis Co., Inc., Gardiner, N.Y.). The mean length (L) is a function of the speed of rotation of the homogenizer blades (omega), time of shearing (t), water concentration ([H2O]), solvent viscosity (eta), temperature (T), and energy of activation (E*), but not a function of the initial length so long as the starting molecules sustain an average of three or more breaks. The relationship of the parameters is expressed by the equation L = (b/omegat1/2eta1/2[H2O])eE*/2kBT, where kB is the Boltzmann constant and b is a constant of proportionality. The breakage rate constant k was determined to have the relationship k = (omega2L2eta[H2O]2/2b2)e-E*/kBT. These equations are valid throughout large ranges of the parameters, and a simple method is described which chooses a final mean length between at least 0.15 and 36 kb by choosing the appropriate shearing conditions and initial fragment length. The heterogeneity of shearing conditions within the shearing vessel permits use of the equations at all breakage rates tested. Based on the work of others using more homogeneous shearing conditions and initial fragment lengths, more complicated forms of the equations are necessary at low breakage rates but not at high ones. A proposed model of the breakage mechanism suggests that molecules with stress-induced localized denaturations break at a rate different from that for native DNA.
D002499:Centrifugation, Density Gradient; D004247:DNA; D006367:HeLa Cells; D008433:Mathematics; D012399:Rotation; D013314:Stress, Mechanical; D014783:Viscosity
D004247:DNA