Synthesis of 3′-O-cyanoethyl-2′-deoxythymidine-5′-monophosphate and its use for the sequencing-by-synthesis technology

For enabling personalized medicine, various attempts have been made for developing a fast and cost-effective DNA-diagnostic method. One of the promising approaches within the last years is the array-based sequencing by synthesis technology. This technique is gel electrophoresis-free and affords a direct read-out of the base sequence via fluorescent signal detection(1). One challenging part of developing this sequencing method is the evolution of a highly tolerable polymerase that incorporates reversible terminators into a DNA-template. Another requirement is the synthesis of a reversible terminator, which is a nucleotide that contains a long fluorescent dye-linker system and a 3'-O-capping group which can be completely removed after incorporation. We found that the cyanoethyl function as 3'-O-blocking group is accepted by the polymerase and also quantitatively cleavable with TBAF in THF.