Biotin-Labeled Oligonucleotides With Extraordinarily Long Tethering Arms

We describe synthesis of four novel biotin phosphoramidites with tethering arms ranging from 20 to 74 atoms in length. One of these phosphoramidites is a uridine derivative with a biotin moiety attached through the 2′-position. The biotin phosphoramidites were synthetized based on robust and efficient methoxyoxalamido (MOX) and succinimido (SUC) precursor strategies from MOX/SUC precursors containing a secondary hydroxyl. They are highly stable in solution (coupling efficiency remains equally high for at least 2 wk after phosphoramidite installation on the synthesizer) and are ideal for the streamlined production of biotin-labeled oligonucleotides. Protocols for the synthesis of biotinylated primers and purification of biotinylated sequencing products by means of streptavidin-coated magnetic beads are also presented.