Enzymatic Synthesis of Polynucleotide Probes

A method of synthesizing a polynucleotide probe having a predetermined sequence and a plurality of labels, the method comprising the steps of:

• a) providing an initiator having a 3′-penultimate deoxyinosine and a 3′-terminal nucleotide having a free 3′ -hydroxyl;
• b) repeating elongation cycles comprising steps of:
• (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′ -hydroxyls, until the polynucleotide probe is formed, wherein (A) at least one of the 3′-O-blocked nucleoside triphosphate is a 3′-O-blocked-(alkyne/azide)-nucleoside triphosphate or a 3′-O-blocked-(protected alkyne/protected azide)-nucleoside triphosphate , (B) in a final elongation cycle a nucleoside triphosphate having a 3′ -alternative blocking group or 3′-O-alternative blocking group is contacted with said elongated fragments having free 3′-hydroxyls, (C) the alternative blocking group has a reactive moiety and may be the same or different than the blocking group, and (D) said step of deblocking is omitted;
• c) reacting a first label having an (alkyne/azide) group with the at least one incorporated 3′ -blocked-(alkyne/azide)-nucleoside triphosphate;
• d) reacting the reactive moiety of the 3′-alternative blocking group or 3′-O-alternative blocking group with a second label having a complementary moiety to form a polynucleotide probe having a plurality of labels;
• and
• e) enzymatically cleaving the polynucleotide probe from the initiator.