Cart
Article
Experimental Design and Polyelectrolyte Effects on Ligand Binding to Nucleic Acids. An Undergraduate Biochemistry Lab
Hi-Res PDFPurchase the full-text
- PDF/HTML,
figures/images,
references and tables,
(where available)
Abstract
We have developed an experiment that utilizes the salt dependence of spermine binding to DNA as a means to introduce experimental design concepts and polyelectrolyte effects in the undergraduate biochemistry lab. The experiment is easily completed in two, three-hour lab periods. Spermine, a multivalent polyamine, competes with ethidium bromide for binding to DNA. Consequently the decrease in the observed ethidium binding constant can be used to determine the equilibrium constant for spermine binding. Binding constants are determined at various concentrations of Na+ and Mg2+ cations in a simple full factorial design. Since cations accumulate near the surface of DNA owing to the polyelectrolyte nature of the nucleic acid, these cations must be displaced by spermine binding. Competition between monovalent and divalent cations for accumulation near the DNA surface results in the two variables being linked; the presence of Mg2+ in the reaction buffer alters how the spermine binding constant changes when the Na+ concentration is altered. Completion of this experiment not only gives students experience with the construction of Scatchard plots but also provides an introduction to experimental design and the importance of polyelectrolyte behavior in nucleic acid chemistry.
Keywords (Audience):
Upper-Division UndergraduateKeywords (Domain):
BiochemistryKeywords (Pedagogy):
Hands-On Learning / ManipulativesKeywords (Subject):
Biophysical ChemistryCiting Articles
Citation data is made available by participants in CrossRef's Cited-by Linking service. For a more comprehensive list of citations to this article, users are encouraged to perform a search in SciFinder.
This article has been cited by 3 ACS Journal articles (3 most recent appear below).
How Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry
Agnieszka Kulczynska, Reed Johnson, Tony Frost, and Lawrence D. MargerumJournal of Chemical Education2011 Article ASAPHow Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry
Agnieszka Kulczynska, Reed Johnson, Tony Frost, and Lawrence D. MargerumJournal of Chemical Education2011 Article ASAPAn advanced undergraduate laboratory project is described that integrates inorganic, analytical, physical, and biochemical techniques to reveal differences in binding between cationic metal complexes and anionic DNA (herring testes). Students were guided ...
Fluorescence Spectroscopy of tRNAPhe Y Base in the Presence of Mg2+ and Small Molecule Ligands
Sarah R. Kirk , Todd P. Silverstein and Karen L. McFarlane HolmanJournal of Chemical Education2008 85 (5), 678Fluorescence Spectroscopy of tRNAPhe Y Base in the Presence of Mg2+ and Small Molecule Ligands
Sarah R. Kirk , Todd P. Silverstein and Karen L. McFarlane HolmanJournal of Chemical Education2008 85 (5), 678This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNAPhe conformational changes induced by ligand binding. In this article we describe a set of ...
Quantitative Determination of DNA–Ligand Binding Using Fluorescence Spectroscopy
Eamonn F. HealyJournal of Chemical Education2007 84 (8), 1304Quantitative Determination of DNA–Ligand Binding Using Fluorescence Spectroscopy
Eamonn F. HealyJournal of Chemical Education2007 84 (8), 1304DAPI, 4′,6-diamidino-2-phenylindole, binds to double-stranded DNA forming a complex that fluoresces up to twenty times more than DAPI alone. It is widely used in biochemical and cytochemical studies for DNA detection. The experiment described here ...
Full Text HTMLTools
-
Add to Favorites
-
Download Citation
-
Email a Colleague -
Permalink
Order Reprints
Rights & Permissions
Citation Alerts
History
- Received: August 03, 2009
ACS
Network
Facebook
Tweet This
CiteULike
Newsvine
Digg This
Delicious
