Showing papers by "Alexander N. Glazer published in 1996"
TL;DR: Energy transfer primers are presented as a new fluorescence labeling paradigm for DNA sequencing and analysis that combines X-ray diffraction and “ghosting” for high-throughput analysis.
Abstract: Energy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis
144 citations
TL;DR: Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phYcobiliproteins, finding that energy transfer on a picosecond time scale does not compete with photodestruction.
Abstract: Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.
127 citations
TL;DR: A novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence is described.
Abstract: DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.
109 citations
TL;DR: Energy transfer (ET) fluorescent primers in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes are described.
82 citations
Patent•
12 Aug 1996TL;DR: In this paper, a spacer consisting of sugar phosphate monomeric units is used for linking to a nucleic acid sequence with any compositions, where an energy transfer component is substituted at the 5' position of the spacer chain.
Abstract: Fluorescent labels are provided employing energy absorber/donor components and energy acceptor/fluorescer components joined together by a spacer which comprises sugar phosphate monomeric units, particularly ribosyl phosphate monomeric units, where the sugar groups are free of active hydroxyl groups. Particularly, an energy transfer component is substituted at the 5' position of the spacer chain, while the other energy transfer component is substituted at the 1' position of the 3' terminal ribosyl group of the label forming an ET cassette for linking to a nucleic acid sequence with any compositions. By employing combinations of ET components, with a common energy absorber/donor and different fluorescers, one can provide for families of labels which can be tagged to any target molecules and which can be excited at a single wavelength and fluoresce at different wavelengths with large Stokes shifts. The compositions find particular application in sequencing.
78 citations
TL;DR: New ET primers are described, with 5- or 6-carboxyrhodamine-6G (G5 or G6) as acceptors (with FAM as the donor) in place of JOE, with improved match in the electrophoretic mobilities of the DNA fragments extended from the ET dye-labeled primers, and less overlap in the fluorescence emission of the various labeled DNA fragments.
52 citations
TL;DR: There is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no Conserved energy transfer pathway common to all native cryptophycean biliproteins.
Abstract: Two crytophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 69 were purified and characterized with respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the α subunit and three on the β subunit. Cr-PC 612 carried phycocyanobilin at α-Cys-18, β-Cys-82, and β-Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at β-DiCys-50,61. Cr-PC 569 carried phycocyanobilin at α-Cys-18 and β-Cys-82, a singly-linked Bilin 584 at β-Cys-158, and a doubly-linked Bilin 584 at β-DiCys-50,61. This work, in conjunction with earlier studies on Cr-PE 545, Cr-PE 555, Cr-PE 566, and Cr-PC 645, shows that there is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no conserved energy transfer pathway common to all native cryptophycean biliproteins. Only phycocyanobilin or phycoerythrobilin is found at β-Cys-82; there is greater bilin variability at the other three attachment sites.
39 citations
TL;DR: The protein and DNA content of the several gel-shifted complexes of HSFDT385-Fl with specific DNA labeled noncovalently with the energy transfer heterodimer TOTAB were accurately determined by a two-color fluorescence emission assay with 488 nm excitation.
26 citations