A RADIATION TARGET METHOD FOR SIZE DETERMINATION OF SUPERCOILED PLASMID DNA

摘要

Supercoiled DNA plasmids were exposed in the frozen state to high energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.