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Nucleic Acids Research, 1988, Vol. 16, No. 11 4937-4956
© 1988

Articles

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes

Mickey S. Urdea, Brian D. Warner, Joyce A. Running, Michelle Stempien, Jennifer Clyne and Thomas Horn

Chiron Corporation 4560 Horton Street, Emeryville, CA 94608, USA

Received January 20, 1988. Revised April 14, 1988. Accepted April 14, 1988.

N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxytrityl-5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 3 2P-labeled probes.


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