RAPID, HIGH-THROUGHPUT DNA SEQUENCING USING CONFOCAL FLUORESCENCE IMAGING OF CAPILLARY ARRAYS

THE GOALS OF THE HUMAN GENOME PROJECT REQUIRE TECHNIQUES FOR DNA SEQUENCING THAT INCREASE THROUGHPUT OVER CURRENT METHODS BY AN ORDER OF MAGNITUDE OR MORE. CURRENT AUTOMATED FLUORESCENCE SEQUENCING INSTRUMENTS REQUIRE 5 TO 10 HOURS FOR A SINGLE GEL RUN AND ACCOMMODATE A MAXIMUM OF 36 REACTION SETS. CAPILLARY GELS CAN BE RUN AT HIGHER VOLTAGES TO YIELD SEPARATION OF 300 TO 500 BASES IN 1 TO 2 HOURS, BUT CURRENT FLUORESCENCE DETECTION METHODS ARE LIMITED TO ANALYZING ONE CAPILLARY AT A TIME. CONFOCAL FLUORESCENCE IMAGING HAS THE CAPACITY TO PRODUCE GREAT SENSITIVITY TO DETECTION, WITH THE GEOMETRICAL ADVANTAGE THAT EXCITATION LIGHT IS DELIVERED THROUGH THE SAME LENS THAT COLLECTS FLUORESCENCE EMISSION. IT HAS RECENTLY BEEN SHOWN THAT A SINGLE SCANNING CONFOCAL FLUORESCENCE DETECTOR CAN DETECT DNA FRAGMENTS IN A PARALLEL ARRAY OF CAPILLARIES. THE FEASIBILITY OF THIS TECHNOLOGY IS BEING TESTED IN THE PHASE I PROJECT FOR HIGH-THROUGHPUT DNA SEQUENCING, INCLUDING AUTOMATED METHODS TO LOAD AND ANALYZE UP TO 200 REACTION SETS SIMULTANEOUSLY IN 1 TO 2 HOURS.