Size Distribution of DNA Molecules Recovered from Non-denaturing Filter Elution

Summary

DNA fragments removed from the filter during non-denaturing filter elution were collected and loaded on top of neutral sucrose gradients. Their size distribution was determined by low-speed centrifugation in neutral sucrose gradients. The average size of eluted DNA was found to be approximately 110 S, yet the average size of DNA collected after short elution times was found to be slightly larger than that after long elution times. It is concluded that the size of eluted DNA fragments is not correlated with their elution rate, and it is proposed that shear forces generated at the pores of the filter cause degradation of the DNA. A comparison of the sedimentation profiles of carefully prepared cellular DNA before and after elution revealed that the generated shear forces during elution break down the DNA to an extent equivalent to around 20 000 DNA double-strand breaks (dsb) per G₁ cell. The size of DNA fragments decreased with increasing radiation dose; however, five times more dsb were found than expected after exposure to radiation alone. It is proposed that this excess of dsb may derive from the transformation of other radiation-induced lesions to dsb under the action of the shear forces generated during elution.