Automated Sequencing with Fluorescent Dyes

As originally developed, both the Maxam-Gilbert and the chain termination methods of DNA sequencing require the use of radioisotopes to visualize the fragments generated in the reactions. However, in addition to being a health risk and presenting a disposal problem, the use of radioactivity makes automation of the DNA sequencing process difficult. Machines that could automatically read DNA sequences did not become practical until fluorescent dyes were introduced as a way to label sequencing fragments.

Two approaches are used to fluorescently label the products of a DNA sequencing reaction: the dye primer method and the dye terminator method. Both are applied only to the chain termination method. The sequencing products made by these methods are electrophoresed on an instrument that uses a laser to detect the different fragments.

In the dye primer method, fluorescent dyes are attached to the 5' ("five prime") end of the primer, which is the end opposite to that where nucleotides are added during chain growth. Four reactions are prepared con-

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