Electrophoresis with Sanger Sequencing

During capillary electrophoresis, the products of the cycle sequencing reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.

A high voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode (Figure 1). Capillary electrophoresis can resolve DNA molecules that differ in molecular weight by only one nucleotide.

Shortly before reaching the positive electrode, the fluorescently labeled DNA fragments, separated by size, move through the path of a laser beam. The laser beam causes the dyes on the fragments to fluoresce. An optical detection device on Applied Biosystems DNA analyzers detects the fluorescence (Figure 2).

The Data Collection Software converts the fluorescence signal to digital data, then records the data in a *.ab1 file. Because each dye emits light at a different wavelength when excited by the laser, all four colors, and therefore, all four bases, can be detected and distinguished in one capillary injection.