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 computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules 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 designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (< 400 nm) from the agarose gel, and by using neutral density filters to prevent saturation of the camera, the fluorescence intensity is linearly related to the amount of DNA varying from approximately 0.03 micrograms in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) to approximately 4 micrograms (where ethidium bromide staining saturates). The percentage DNA released from the plug and distribution in the lane (with 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of radioactivity of the same DNA labeled with [3H]dThd. However, scattering 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 problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for approximately 7% of the fluorescence from the plug tailing out approximately 10 mm beyond the first 1 mm in the lane. In addition, 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 migration stops.
Dewey
W C
WC
Radiation Oncology Research Laboratory, University of California, San Francisco 94103.
Thompson
L L
LL
Trinh
M L
ML
Latz
D L
DL
Ward
J F
JF
D004247:DNA / Q000032:analysis*; D016521:Electrophoresis, Gel, Pulsed-Field; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted
D004247:DNA