DNA-SEQUENCING BY CAPILLARY ELECTROPHORESIS WITH REPLACEABLE LINEAR POLYACRYLAMIDE AND LASER-INDUCED FLUORESCENCE DETECTION

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficul ties associated with cross-linked polyacrylamide gel stability have be en overcome for the routine application of CE to DNA sequencing. A sim ple laser-induced fluorescence (LIF) detection system based on a singl e laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluo rescent dyes and two peak height ratios, detected in two optical chann els. FAM- and JOE-labeled M13 (-21) primers have been chosen because b oth dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA f ragment mobilities. Addition of denaturants to the electrophoresis run ning buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32-degrees-C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymeri zation reaction of LPA has led to methodology that has proven to be re producible for obtaining DNA sequencing information of M13mp18 phage f or 350 nucleotides in close to 30 min.