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Nucleic Acids Research, Vol 26, Issue 4 1026-1031, Copyright © 1998 by Oxford University Press

ARTICLES

Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay

B Mullah, K Livak, A Andrus and P Kenney
PE Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. mullahbk@perkin-elmer.com

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye- labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t- butylamine:methanol:water is used for cleavage and deprotection of dye- labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.
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