Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides

Abstract

Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.