Academic Journal
Binding, brightness, or noise? Extracting temperature-dependent properties of dye bound to DNA.
| Τίτλος: | Binding, brightness, or noise? Extracting temperature-dependent properties of dye bound to DNA. |
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| Συγγραφείς: | DeJaco RF; Applied and Computational Mathematics Division, National Institute of Standards and Technology, Gaithersburg, Maryland; Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland. Electronic address: robert.dejaco@nist.gov., Majikes JM; Microsystems and Nanotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland., Liddle JA; Microsystems and Nanotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland., Kearsley AJ; Applied and Computational Mathematics Division, National Institute of Standards and Technology, Gaithersburg, Maryland. |
| Πηγή: | Biophysical journal [Biophys J] 2023 Apr 04; Vol. 122 (7), pp. 1364-1375. Date of Electronic Publication: 2023 Mar 04. |
| Τύπος έκδοσης: | Journal Article; Research Support, Non-U.S. Gov't |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Cell Press Country of Publication: United States NLM ID: 0370626 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1542-0086 (Electronic) Linking ISSN: 00063495 NLM ISO Abbreviation: Biophys J Subsets: MEDLINE |
| Imprint Name(s): | Publication: Cambridge, MA : Cell Press Original Publication: New York, Published by Rockefeller University Press [etc.] for the Biophysical Society. |
| Ιατρικοί όροι (MeSH): | DNA*/chemistry , DNA, Single-Stranded*, Fluorescent Dyes/chemistry ; Temperature ; Organic Chemicals |
| Περίληψη: | We present a method for extracting temperature-dependent thermodynamic and photophysical properties of SYTO-13 dye bound to DNA from fluorescence measurements. Together, mathematical modeling, control experiments, and numerical optimization enable dye binding strength, dye brightness, and experimental noise (or error) to be discriminated from one another. By focusing on the low-dye-coverage regime, the model avoids bias and can simplify quantification. Utilizing the temperature-cycling capabilities and multi-reaction chambers of a real-time PCR machine increases throughput. Significant well-to-well and plate-to-plate variation is quantified by using total least squares to account for error in both fluorescence and nominal dye concentration. Properties computed independently for single-stranded DNA and double-stranded DNA by numerical optimization are consistent with intuition and explain the advantageous performance of SYTO-13 in high-resolution melting and real-time PCR assays. Distinguishing between binding, brightness, and noise also clarifies the mechanism for the increased fluorescence of dye in a solution of double-stranded DNA compared to single-stranded DNA; in fact, the explanation changes with temperature. (Published by Elsevier Inc.) |
| Competing Interests: | Declaration of interests Certain commercial equipment, instruments, software, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose. |
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| Substance Nomenclature: | 0 (DNA, Single-Stranded) 9007-49-2 (DNA) 0 (SYTO 13) 0 (Organic Chemicals) 0 (Fluorescent Dyes) |
| Entry Date(s): | Date Created: 20230305 Date Completed: 20230407 Latest Revision: 20240405 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC10111365 |
| DOI: | 10.1016/j.bpj.2023.03.002 |
| PMID: | 36871160 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1542-0086 |
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| DOI: | 10.1016/j.bpj.2023.03.002 |