EFFICIENT SYNTHESIS OF DOUBLE DYE-LABELED OLIGODEOXYRIBONUCLEOTIDE PROBES AND THEIR APPLICATION IN A REAL-TIME PCR ASSAY

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled suppo rt has been developed for automated synthesis of double dye-labeled ol igodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylami ne: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 i s significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage, These double dye-labeled prob es 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 performan ce of probes synthesized using the dye-labeled support compared with t raditional post-synthetic attachment of rhodamine.