3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing

A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

• (i) attaching the 5′-end of a primer to a solid surface;
• (ii) hybridizing a target nucleic acid to the primer attached to the solid surface to form a hybridized primer/target nucleic acid complex;
• (iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters:
• wherein:
• R1 is triphosphate or α-thiotriphosphate;
• R2 is hydrogen or hydroxy;
• R3 is alkyl(C≦8) or substituted alkyl(C≦8);
• R4 is
• hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
• alkyl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6), amido(C≦6), or a substituted version of any of these groups;
• R5 is a -linker-reporter, wherein the reporter is based on a dye;
• R6 is:
• hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
• alkyl(C≦6), alkenyl(C≦6), alkynyl(C≦6), aryl(C≦6), aralkyl(C≦8), heteroaryl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6), amido(C≦6), or a substituted version of any of these groups;
• or a salt, tautomer, or optical isomer thereof;
• (iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction;
• (v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its reporter;
• (vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula:
• with the variables as defined by the claim referenced in step (iii);
• and
• (vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.

A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

• (i) attaching the 5′-end of a target nucleic acid to a solid surface;
• (ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a hybridized primer/target nucleic acid complex;
• (iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters:
• wherein:
• R1 is triphosphate or α-thiotriphosphate;
• R2 is hydrogen or hydroxy;
• R3 is alkyl(C≦8) or substituted alkyl(C≦8);
• R4 is
• hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
• alkyl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6) amido(C≦6), or a substituted version of any of these groups;
• R5 is a -linker-reporter, wherein the reporter is based on a dye;
• R6 is:
• hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
• alkyl(C≦6), alkenyl(C≦6), alkynyl(C≦6), aryl(C≦6), aralkyl(C≦8), heteroaryl(C≦6), acyl(C≦6), alkoxy(C≦6), acyloxy(C≦6), alkylamino(C≦6), dialkylamino(C≦6), amido(C≦6), or a substituted version of any of these groups;
• or a salt, tautomer, or optical isomer thereof;
• or a salt, tautomer, or optical isomer thereof;
• (iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction;
• (v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its reporter;
• (vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula:
• with the variables as defined by the claim referenced in step (iii);
• and
• (vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.