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Nonisotopic DNA Detection System Employing Elastase and a Fluorogenic Rhodamine Substrate

Johnson, Arthur F. and Struthers, Mary D. and Pierson, Keith B. and Mangel, Walter F. and Smith, Lloyd M. (1993) Nonisotopic DNA Detection System Employing Elastase and a Fluorogenic Rhodamine Substrate. Analytical Chemistry, 65 (17). pp. 2352-2359. ISSN 0003-2700.

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An alternative fluorescence-based method has been developed for the direct detection of small quantities of DNA in solution. In this system, a serine protease (elastase) is coupled to a DNA oligonucleotide through a disulfide linkage. A bis(tetraalanine)-derivatized rhodamine molecule (BZTAlaR) has been synthesized for use as a substrate. BZTAlaR is nonfluorescent in its derivatized form and shows negligible hydrolysis in solution. Cleavage of the tetraalanyl groups from the rhodamine portion of the molecule restores its fluorescence. Hybridization of the elastase-oligonucleotide conjugate to its target, capture of the conjugate-target complex with streptavidin-coated magnetic beads, addition of substrate, and subsequent detection of the target by fluorescence are accomplished in solution. Hybridization is rapid and specific, with over 90% of a target sequence successfully hybridized and captured. This method exhibits low background and an amplified fluorescent signal over time, resulting in a current detection limit of 0.49 fmol of elastase alone, or 2.64 fmol of conjugate, within 2 h.

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Additional Information:© 1993 American Chemical Society. Published in print 1 September 1993. Received for review March 16, 1993. Accepted May 26, 1993. We thank Dr. Michael Hunkapiller for suggesting elastase as a more stable alternative to trypsin, Tom Wallow for synthesis of the 5‘-monomethoxytrityl phosphoramidite, and Professor M. Thomas Record for the use of the SLM8000C spectrofluorometer. This work was supported by NSF Grant DIR 8957582 and Baxter Healthcare Corp.
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Baxter Healthcare CorporationUNSPECIFIED
Issue or Number:17
Record Number:CaltechAUTHORS:20180130-081252773
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Official Citation:Nonisotopic DNA detection system employing elastase and a fluorogenic rhodamine substrate Arthur F. Johnson, Mary D. Struthers, Keith B. Pierson, Walter F. Mangel, and Lloyd M. Smith Analytical Chemistry 1993 65 (17), 2352-2359 DOI: 10.1021/ac00065a031
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84569
Deposited By: Ruth Sustaita
Deposited On:31 Jan 2018 18:45
Last Modified:03 Oct 2019 19:20

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