Single-Nucleotide Primer Extension Assay by Capillary Electrophoresis Laser-Induced Fluorescence

One of the most sensitive methods of detection for capillary electrophoresis (CE) is laser-induced fluorescence (LIF). The reader is referred to a recent review (1) for a more detailed description of the technique, but in brief, laser irradiation is used to excite the fluorescent molecule, and the emission is detected at a distinct wavelength. Since most molecules do not exude native fluorescence, analytes can be derivatized with one of several commercially available fluorescent dyes either before, after or during the separation step. This leads to the major advantage of LIF, the low background and subsequent sensitivity, which affords a factor of 1000 or more improvement in detection limit compared to UV absorbance. One of the most popular applications of CE-LIF is the analysis of DNA either by specifically labeling one or more nucleotides with a fluorescent tag (e.g., on the primer or the dideoxyterminator) or nonspecifically detecting DNA through intercalation or similar binding of dyes (2).