AN IMPROVED IMAGING SYSTEM USING A CCD DETECTOR TO QUANTITATE FLUORESCENCE FROM STAINED DNA GELS

THE MASS OF DNA SEPARATED BY GEL ELECTROPHORESIS CAN BE DETERMINED FROM THE INTENSITY OF THE FLUORESCENCE OF A DNA-SPECIFIC DYE EXCITED BY UV RADIATION. THE PRESENT USE OF PHOTOGRAPHIC FILM TO RECORD THE FLUORESCENCE LIMITS THE ACCURACY, SENSITIVITY, AND DYNAMIC RANGE OF THE ANALYSIS. ALTERNATE SCANNING SYSTEMS THAT DIRECTLY DETECT THE FLUORESCENCE ARE SLOW. A NEW IMAGING SYSTEM IS PLANNED TO MEASURE BOTH THE MASS AND THE MOLECULAR WEIGHT OF THE SAMPLE. SUBSTANTIAL INSTRUMENTAL IMPROVEMENTS APPEAR POSSIBLE IN FLUORESCENT EXCITATION, OPTICAL LIGHT COLLECTION AND IMAGING, AND DETECTOR PERFORMANCE. REPLACINGFILM WITH A CHARGE-COUPLED ARRAY DETECTOR (CCD) NOW APPEARS ATTRACTIVE BECAUSE OF RECENT IMPROVEMENTS IN CCD PERFORMANCEAND THE AVAILABILITY OF LARGER ARRAYS AT LOWER COST. THOSE OPTICAL PROPERTIES OF THE STAINED DNA/GEL SYSTEM WILL BE MEASURED, WHICH MUST BE WELL-CHARACTERIZED TO DESIGN A HIGH PERFORMANCE IMAGING SYSTEM AND TO ESTIMATE ITS PERFORMANCE WITH USEFUL ACCURACY. AN ENGINEERING ANALYSIS OF A SPECIFIC DESIGN WILL EXAMINE THE INCREASED ANALYTICAL ABILITY TO QUANTITATE THE MASS OF DNA IN A BAND THROUGH IMPROVED FLUORESCENCE MEASUREMENT, AND WHETHER THE OVERALL MEASUREMENT TIME CAN BE SIGNIFICANTLY SHORTENED BY ACHIEVING HIGH DETECTOR SIGNAL-TO-NOISE AND BY MORE FULLY AUTOMATING THE MEASUREMENT. THE PHASE I STUDY WILL ADDRESS THE FEASIBILITY OF THE NEW INSTRUMENTAL APPROACH, ESTIMATE THE LIMITS OF PERFORMANCE, AND PROVIDE A DESIGN DIRECTION FOR THE PROTOTYPE HARDWARE TO FOLLOW IN PHASE II.