Finding the optimal conformation for fluorescent labeled nucleosides is difficult for standard GAs. We investigated the structure of molecule and of the resulting search space itself and concluded that the problem is of the “needle in the haystack” type. Using niching and island models and a crossover operator adapted to the underlying problem, the GA was able to find the global optimum. Each of these improvements increased the probability to find the global optimum from 0

A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.

Biomedical ChromatographyVolume 13, Issue 2 p. 109-110 Extended Abstract Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures M. Neumann, Corresponding Author M. Neumann Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanyPhysikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorW. Ehrfeld, W. Ehrfeld Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorK.-T. Han, K.-T. Han Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorP. Jacob, P. Jacob Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorR. Konrad, R. Konrad Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorS. Siebert, S. Siebert Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorJ. Wolfrum, J. Wolfrum Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorM. Sauer, M. Sauer Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this author M. Neumann, Corresponding Author M. Neumann Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanyPhysikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorW. Ehrfeld, W. Ehrfeld Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorK.-T. Han, K.-T. Han Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorP. Jacob, P. Jacob Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorR. Konrad, R. Konrad Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Straße 18–20, 55129 Mainz, GermanySearch for more papers by this authorS. Siebert, S. Siebert Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorJ. Wolfrum, J. Wolfrum Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this authorM. Sauer, M. Sauer Physikalisch-Chemisches-Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, GermanySearch for more papers by this author First published: 30 April 1999 https://doi.org/10.1002/(SICI)1099-0801(199904)13:2<109::AID-BMC854>3.0.CO;2-RCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume13, Issue2Special Issue: Extended Abstracts from VIIIth International Symposium on Luminescence Spectrometry in Biomedical and Environmental Analysis – Detection Techniques and Applications in Chromatography and Capillary Electropharesis. 26–29 May 1998. Held at University of Las Palmas de Gran Canaria, Canary Islands, SpainApril 1999Pages 109-110 RelatedInformation

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.

The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biological applications with absorption and emission beyond 600 nm. We applied the pattern recognition technique taken from information theory to ultrasensitive fluorescence detection and identification of dye molecules. Using pulsed diode lasers emitting at 630-640 nm in combination with new efficient rhodamine and oxazine dyes four-dye one-lane DNA sequencing in capillary gelelectrophoresis with time-resolved fluorescence detection is demonstrated.Applying a confocal fluorescence microscope the detection sensitivity could be further increased allowing the time-resolved fluorescence detection and identification of individual labeled analyte molecules in water.

A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5′-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.

We applied a short-pulse diode laser emitting at 637 nm with a repetition rate of 30 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual labeled mononucleotide molecules in water. A newly synthesized oxazine dye (MR121) and the commercially available carbocyanine dye Cy5 were used as fluorescent labels. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals of the two mononucleotides were analyzed and identified by a maximum likelihood estimator. The results showed out that 60 detected photons per transit of a single molecule are sufficient to distinguish two labeled mononucleotides in water with a misclassification of less than 10% via their characteristic fluorescence lifetimes of 1.07+/-0.27 ns (Cy5-dUTP) and 1.89+/-0.34 ns (MR121-dUTP).

We developed a simple, tiny setup with only a few optical devices for fast and sensitive identification of DNA with a short-pulse semiconductor laser operating at 20 MHz. In combination with newly synthesized fluorescent dyes (rhodamine derivatives) which exhibit high fluorescence quantum efficiencies and distinct fluorescence lifetimes at semiconductor laser excitation wavelength a sensitive detection and time-resolved identification of DNA can be achieved. At an excitation wavelength of 635 nm the fluorescence background is greatly reduced. We demonstrate the DNA identification of A- and G-terminated DNA fragments labeled at the 5'-end with the rhodamine derivatives MR 200- 1 and JA 169 during capillary gel electrophoresis. The characteristic time-resolved data are acquired by the time- correlated single-photon counting technique. Time-resolved identification analysis is realized by the maximum likelihood estimator. For prediction of the error rate (misclassification) Cramers equation in combination with a pattern recognition technique is applied. These methods deliver high reliabilities at low classification error rates for low fluorescence light level applications.

We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).