PloS one, 2017-04, Vol.12 (4), p.e0175766-e0175766
2017
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Details
- Autor(en) / Beteiligte
- Titel
- Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules
- Ist Teil von
- PloS one, 2017-04, Vol.12 (4), p.e0175766-e0175766
- Ort / Verlag
- United States: Public Library of Science
- Erscheinungsjahr
- 2017
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1932-6203eISSN: 1932-6203DOI: 10.1371/journal.pone.0175766Titel-ID: cdi_plos_journals_1889361413
- Format
- –
- Schlagworte
- Addition polymerization, Adhesion, Arrays, Artificial intelligence, Assaying, Bacteria, Binding energy, Biochemistry, Biology, Biology and life sciences, Chemical compounds, Chemistry, Color, Computer and Information Sciences, Data acquisition, Data collection, Data processing, Deoxyribonucleic acid, Design analysis, Diffraction, Diffusion, DNA, DNA - analysis, DNA-directed RNA polymerase, DNA-Directed RNA Polymerases - metabolism, E coli, Efficiency, Elongation, Energy transfer, Engineering and Technology, Equipment Design, Escherichia coli - enzymology, Escherichia coli - genetics, Excitation, Experiments, Fluorescence, Fluorescence microscopy, Fluorescence resonance energy transfer, Fluorescence spectroscopy, Fluorescent indicators, Fluorophores, Geometry, Grants, High-Throughput Screening Assays - instrumentation, Intellectual property, Kinetics, Lasers, Melting, Methods, Microscopy - instrumentation, Microscopy, Confocal - instrumentation, Molecular biology, Optics, Physical Sciences, Promoter Regions, Genetic, Protein folding, Research and Analysis Methods, Resonance, Ribonucleic acid, RNA, RNA polymerase, Silicon, Spectrometry, Fluorescence - instrumentation, Statistical analysis, Studies, Technology, Technology utilization, Theoretical analysis, Time, Tracking, Transcription (Genetics), Transcription initiation, Transcription, Genetic, Translocation, Waveguides