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ERIC Number: EJ1090612
Record Type: Journal
Publication Date: 2016-Feb
Pages: 4
Abstractor: As Provided
ISBN: N/A
ISSN: ISSN-0021-9584
EISSN: N/A
Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity
Pazhani, Yogitha; Horn, Abigail E.; Grado, Lizbeth; Kugel, Jennifer F.
Journal of Chemical Education, v93 n2 p383-386 Feb 2016
FRET (Fo¨rster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students measured the apparent FRET between donor and acceptor fluorophores located on the ends of several DNAs of unknown lengths, enabling them to order the DNAs according to size. In addition, students investigated site-specific DNA cleavage by restriction endonucleases, using loss of apparent FRET to determine which enzyme cut sites were present in each of the DNAs. After completing this experiment, students understood the inverse relationship between changes in FRET and changes in distance, and understood how changes in FRET could be used to monitor a conformational change in a molecule. As an extension to the experiment, a tutorial is included that uses the same DNAs to illustrate the ability of single-molecule FRET measurements to resolve heterogeneity in a sample, which cannot be done via more traditional ensemble measurements.
Division of Chemical Education, Inc and ACS Publications Division of the American Chemical Society. 1155 Sixteenth Street NW, Washington, DC 20036. Tel: 800-227-5558; Tel: 202-872-4600; e-mail: eic@jce.acs.org; Web site: http://pubs.acs.org/jchemeduc
Publication Type: Journal Articles; Reports - Descriptive
Education Level: Higher Education; Postsecondary Education
Audience: N/A
Language: English
Sponsor: National Science Foundation (NSF)
Authoring Institution: N/A
Grant or Contract Numbers: MCB1244518