A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished 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 fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide 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, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

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We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP).

We applied a short‐pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora‐diaza‐indacene dye (Bodipy630/650), were used as fluorescent labels. The time‐resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5‐dCTP and Bodipy‐dUTP, can be identified by time‐resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time‐resolved identification of Cy5‐dCTP, MR121‐dUTP and Bodipy‐dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5‐dCTP), 2.07 ± 0.59 ns (MR121‐dUTP) and 3.88 ± 1.71 ns (Bodipy‐dUTP).

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