Broker, Thomas R. and Angerer, Lynne M. and Yen, Pauline H. and Hershey, N. Davis and Davidson, Norman (1978) Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labels. Nucleic Acids Research, 5 (2). pp. 363-384. ISSN 0305-1048 http://resolver.caltech.edu/CaltechAUTHORS:BROnar78
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A method is described for indirect electron microscopic visualization and mapping of tRNA and other short transcripts hybridized to DNA. This method depends upon the attachment of the electron-dense protein ferritin to the RNA, the binding being mediated by the remarkably strong association of the egg white protein avidin with biotin. Biotin is covalently attached to the 3' end of tRNA using an NH2 (CH2) 5NH2 bridge. The tRNA-biotin adduct is hybridized to complementcrry DNA sequences present in a single stranded nonhomology loop of a DNA:DNA heteroduplex. Avidin, covalently crosslinked to ferritin is mixed with the heteroduplex and becomes bound to the hybridized tRNA-biotin. Observation of the DNA:RNA-biotin:avidin-ferritin complex by electron microsdopy specifically and accurately reveals the position of the tRNA gene, with a frequency of labeling of approximately 50%.
|Additional Information:||Copyright © 1978 Oxford University Press. Received October 31, 1977. We gratefully acknowledge our colleagues Drs. Louise T. Chow, Maria Pellegrini, and Chin Hua Wu for helpful discussions and experimental contributions to the development of these techniques. TRB was supported by a Helen Hay Whitney Fellowship, LMA by a Damon Runyan Fellowship, and NDH by an NSF Fellowship. General research support was received from an NIH grant to ND.|
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|Deposited By:||Tony Diaz|
|Deposited On:||25 Jul 2006|
|Last Modified:||26 Dec 2012 08:57|
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