MULTIPLEX DYE DNA-SEQUENCING IN CAPILLARY GEL-ELECTROPHORESIS BY DIODE LASER-BASED TIME-RESOLVED FLUORESCENCE DETECTION

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently l abeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA24 2), and a commercially available cyanine dye (CY5). The dye fluorescen ce was excited by a pulsed diode laser emitting at 630 nm. The fluores cence decay was detected by an avalanche photodiode using a single-fil ter system. The dyes used here, so-called multiplex dyes, can be disti nguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA frag ments, capillary gel electrophoresis (CGE) with a 5% linear polyacryla mide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length, First, time-resolved, on e-lane, four-dye DNA sequencing runs in CGE are presented. The sequenc e information of 660 bp was determined with a probability of correct c lassification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with o ther methods.