MODIFICATIONS OF ALKALINE MICROGEL ELECTROPHORESIS FOR SENSITIVE DETECTION OF DNA-DAMAGE

The alkaline microgel electrophoresis technique was modified to achiev e a substantial increase in sensitivity for the detection of radiation -induced DNA damage in human lymphocytes. This increased sensitivity w as achieved through: (1) the addition of free radical scavengers to th e electrophoresis solution to reduce DNA damage generated during alkal ine unwinding and electrophoresis; (2) the modification of the electro phoresis unit to achieve a more uniform electric field; (3) the use of YOYO-1, a DNA dye, producing fluorescence 500-fold more intense than ethidium bromide; and (4) the introduction of an image analysis system for the quantitation of DNA migration. In addition to increasing sens itivity, these modifica tions have increased the speed with which obse rvations can be quantified, and improved reproducibility from experime nt to experiment. In human lymphocytes, these modifications have resul ted in an increased sensitivity of several fold, allowing the detectio n of DNA damage in the range of 50 mGy. This increased sensitivity for the detection of DNA damage should extend the utility of this techniq ue.