DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser-induced fluorescence detection

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Abstract

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running 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 polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.

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