On-the-fly fluorescence lifetime detection of labeled DNA primers

Li, Liang-Chi; He, Hui; Nunnally, Brian K.; McGown, Linda B.

doi:10.1016/s0378-4347(97)00102-3

Cited by 31 publications

(24 citation statements)

References 12 publications

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“…Shown are the 5-isomer for FAM and the 6-isomer for JOE, TAMRA, and ROX. tiple dyes having overlapping spectral emission can be distinguished (CHANG et al, 1993;HAN et al, 1993;SAUER et al, 1994;LEGENDRE et al, 1996;SOPER et al, 1996;MULLER et al, 1997;NUNNALLY et al, 1997;FLANAGAN et al, 1998). Methods for "on-the-fly'' lifetime measurements of labeled DNA fragments have been described for capillary electrophoresis …”

Section: Fluorescence Lifetimementioning

confidence: 99%

“…The first type uses the one-dye/four-lane approach in which the identity of the chainterminating nucleotide at each position is determined by running four separate reactions each of which contains the same fluorescent embodiments (LI and MCGOWN, 1996; LI et al, 1997) and slab gel embodiments (LASSITER et al, 2000). Besides enabling common excitation, fluorescence lifetime discrimination also permits the use of common spectral detection optics.…”

Section: Biochemistry Of Dna Sequencingmentioning

confidence: 99%

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Sequencing Technology

et al. 2001

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Section: Fluorescence Lifetimementioning

confidence: 99%

Section: Biochemistry Of Dna Sequencingmentioning

confidence: 99%

Sequencing Technology

et al. 2001

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“…Several groups have been investigating the potential use of fluorescence lifetime discrimination techniques for identifying nucleotide bases in DNA sequencing using both time-resolved [1][2][3][4][5][6] and phase-resolved methods [7][8][9][10][11][12]. Wolfrum and co-workers [13] recently demonstrated the use of a four-lifetime approach to calling bases in DNA sequencing using capillary gel electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

et al. 2002

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We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near-infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye-primer versus dye-terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross-linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near-IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye-terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye-primer within the gel lane.

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“…When DNA sequencing is carried out, four different dyes, one for each of the four nucleotide bases, are typically analyzed in separate detection channels. Efforts directed toward increased throughput has resulted in studies focused on the use of fluorescence lifetime discrimination techniques by several research groups using either time-resolved 1-5 or phase-resolved [6][7][8][9] methods to expand on the multiplexing capabilities of fluorescence. The use of a four-lifetime detection concept to calling bases in DNA sequencing has recently been described by Wolfrum et al 10 .…”

Section: Introductionmentioning

confidence: 99%

<title>Multiplexed analysis using time-resolved near-IR fluorescence for the detection of genomic material</title>

et al. 2002

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While fluorescence continues to be an important tool in genomics, new challenges are being encountered due to increased efforts toward miniaturization reducing detection volumes and the need for screening multiple targets simultaneously. We have initiated work on developing time-resolved near-IR fluorescence as an additional tool for the multiplexed analyses of DNA, either for sequencing or mutation detection. We have fabricated simple and compact time-resolved fluorescence microscopes for reading fluorescence from electrophoresis or DNA microarrays. These microscopes consist of solid-state diode lasers and diode detectors (SPADs) and due to their compact size, the optical components and laser head can be mounted on high-speed micro-translational stages to read fluorescence from either multi-channel capillary electrophoresis instruments or microfabricated DNA sorting devices. The detector is configured in a time-correlated single photon counting format to allow acquisition of fluorescence lifetimes on-the-fly during data acquisition in the limit of low counting statistics. In multiplexed analyses, lifetime discrimination serves as a method for dye-reporter identification using only a single readout channel. Also, coupled to multi-color systems, lifetime identification can significantly increase the number of probes monitored in a single instrument. In this work, near-IR fluorescence, including dye-labels and hardware, will be discussed as well as the implementation of near-IR fluorescence in DNA sequencing using slab gel electrophoresis and DNA microarrays.

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On-the-fly fluorescence lifetime detection of labeled DNA primers

Cited by 31 publications

References 12 publications

Sequencing Technology

Sequencing Technology

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

<title>Multiplexed analysis using time-resolved near-IR fluorescence for the detection of genomic material</title>

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