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.
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