Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Single molecule DNA sequencing in submicrometer channels: state of the artand future prospects
Autore:
Sauer, M; Angerer, B; Ankenbauer, W; Foldes-Papp, Z; Gobel, F; Han, KT; Rigler, R; Schulz, A; Wolfrum, J; Zander, C;
Indirizzi:
Univ Heidelberg, Inst Phys Chem, D-69120 Heidelberg, Germany Univ Heidelberg Heidelberg Germany D-69120 , D-69120 Heidelberg, Germany Roche Mol Biochem, D-82377 Penzberg, Germany Roche Mol Biochem Penzberg Germany D-82377 em, D-82377 Penzberg, Germany Univ Gesamthsch Siegen, Inst Phys Chem, D-57068 Siegen, Germany Univ Gesamthsch Siegen Siegen Germany D-57068 m, D-57068 Siegen, Germany Karolinska Inst, Dept Med Biophys, S-17177 Stockholm, Sweden Karolinska Inst Stockholm Sweden S-17177 phys, S-17177 Stockholm, Sweden
Titolo Testata:
JOURNAL OF BIOTECHNOLOGY
fascicolo: 3, volume: 86, anno: 2001,
pagine: 181 - 201
SICI:
0168-1656(20010413)86:3<181:SMDSIS>2.0.ZU;2-U
Fonte:
ISI
Lingua:
ENG
Soggetto:
FLUORESCENCE CORRELATION SPECTROSCOPY; TIME-RESOLVED IDENTIFICATION; LASER-INDUCED FLUORESCENCE; AQUEOUS-SOLUTION; DIODE-LASER; MICROSTRUCTURES; BIOTECHNOLOGY; LIQUIDS; PROBES;
Keywords:
single-molecule DNA sequencing; time-resolved fluorescence detection; diode lasers; submicrometer channels and electrophoresis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
48
Recensione:
Indirizzi per estratti:
Indirizzo: Sauer, M Univ Heidelberg, Inst Phys Chem, Neuenheimer Feld 253, D-69120 Heidelberg,Germany Univ Heidelberg Neuenheimer Feld 253 Heidelberg Germany D-69120
Citazione:
M. Sauer et al., "Single molecule DNA sequencing in submicrometer channels: state of the artand future prospects", J BIOTECH, 86(3), 2001, pp. 181-201

Abstract

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 mum. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed similar to 50 mum in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43 +/-0.19 ns (Cy5-dCMP), and 2.35 /-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA. (C) 2001 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/09/20 alle ore 06:06:16