A CHARGE-COUPLED-DEVICE CAMERA IMAGE-ANALYSIS SYSTEM FOR QUANTIFYING DNA DISTRIBUTIONS IN AGAROSE GELS AFTER PULSED-FIELD GEL-ELECTROPHORESIS

A charge-coupled-device camera system was coupled to a personal comput er and, with uniformity in illumination and detection (within 4-8%) al ong each lane, was used for quantifying the distribution of DNA molecu les that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially desi gned transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescen ce (<400 nm) from the agarose gel, and by using neutral density filter s to prevent saturation of the camera, the fluorescence intensity is l inearly related to the amount of DNA varying from similar to 0.03 mu g in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) t o similar to 4 mu g (where ethidium bromide staining saturates). The p ercentage DNA released from the plug and distribution in the lane (wit h 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of ra dioactivity of the same DNA labeled with [H-3]dThd. However, scatterin g of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This pr oblem was overcome by using a form with slots to cover every other lan e when the images were obtained and either (1) cutting the lane from t he plug and moving it 15 mm away or (2) imaging the intact gel and app lying a correction for similar to 7% of the fluorescence from the plug tailing out similar to 10 mm beyond the first 1 mm in the lane. In ad dition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA mi gration stops.