Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps

1. A method to determine the identity of a nucleotide residue in an extension product, comprising:

(a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, at least 4 distinguishable, blocked deoxyribonucleotide triphosphate (dNTP) analogue species, at least 3 of which distinguishable, blocked dNTP analogue species are labeled such that each distinguishable, blocked dNTP analogue species in the sequencing reaction mixture can be distinguished from the other distinguishable, blocked dNTP analogue species, thereby incorporating a distinguishable, blocked dNTP analogue species into at least one of said plurality of identical primed template DNA molecules and forming a distinguishable, blocked extension product; and

(b) after step (a), removing unincorporated distinguishable, blocked dNTP analogues followed by adding to the sequencing reaction mixture at least 4 unlabeled, blocked dNTP analogue species, incorporating an unlabeled, blocked dNTP analogue species into at least one of said plurality of identical primed template DNA molecules thereby forming an unlabeled, blocked extension product; and

(c) upon addition of the at least 4 unlabeled, blocked dNTP analogue species during step (b), determining the identity of the distinguishable, blocked dNTP analogue incorporated into the distinguishable, blocked extension product in step (a), wherein the combined steps (a)-(c) occurs for about 1 minute to about 10 minutes.

2. The method according to claim 1 further comprising assigning the complement of the distinguishable, blocked dNTP analogue determined in step (c) as the identity of the nucleotide residue in the template DNA molecule.

3. The method according to claim 2 comprising iterating steps (a)-(c) one or more times, thereby determining a nucleotide sequence of at least a portion of the template DNA molecule.

4. The method according to claim 1 that, after step (c), further comprises (d) chemical, enzymatic, or photoactivated cleavage, of a blocking moiety linked to a 3′-oxygen atom of a terminal deoxyribose of the distinguishable, blocked extension products and/or unlabeled, blocked extension products to generate extensible extension products.

5. The method according to claim 1 that comprises iterating steps (a)-(c) one or more times, wherein after step (c) of each iteration, the distinguishable, blocked extension products and unlabeled, blocked extension products are treated to generate extendible extension products, with the proviso that after the last iteration, treatment of distinguishable, blocked extension products and unlabeled, blocked extension products can be omitted.

6. The method according to claim 1 wherein the labeled, 3′-blocked dNTP analogues are:

(i) a labeled, 3′-blocked dGTP analogue;

(ii) a labeled, 3′-blocked dCTP analogue;

(iii) a labeled, 3′-blocked dATP analogue; and

(iv) a labeled, 3′-blocked dTTP analogue or a labeled, 3′-blocked dUTP analogue;

wherein the labeled, 3′-blocked dGTP, dCTP, dATP, dTTP, and/or dUTP analogue(s) comprise:

an incorporable nucleoside or incorporable analogue thereof; the labeled, 3′-blocked dGTP, dCTP, dATP, dTTP, and/or dUTP analogue(s) comprise a sugar moiety that is deoxyribose or a deoxyribose analogue;

a cleavable blocking moiety, wherein the cleavable blocking moiety optionally is linked to the 3′-position of the sugar moiety, wherein the sugar moiety optionally is deoxyribose or a deoxyribose analogue; and

a unique label molecule that differs from the unique label molecules of each of the other labeled, 3′-blocked dNTP analogue species such that each of the labeled, 3′-blocked dNTP analogue species is distinguishable from the other labeled, 3′-blocked dNTP analogue species.

7. The method of claim 6 , wherein the label molecules are selected from the group consisting of a dye, a chromophore, a combinatorial fluorescence energy transfer label, an electrophore, a fluorophore, a mass label, and a radiolabel.

8. The method according to claim 6 wherein the cleavable blocking moiety of the labeled, 3′-blocked dNTP analogues is linked to the 3′-oxygen of a deoxyribose via an allyl moiety, a 2-nitrobenzyl moiety, an azido moiety, or a disulfide moiety.

9. The method according to claim 1 wherein the unlabeled, 3′-blocked dNTP analogue species are:

(i) an unlabeled, 3′-blocked dGTP;

(ii) an unlabeled, 3′-blocked dCTP;

(iii) an unlabeled, 3′-blocked dATP; and

(iv) an unlabeled, 3′-blocked dTTP or an unlabeled, 3′-blocked dUTP;

wherein the unlabeled, 3′-blocked dNTPs comprise:

an incorporable nucleoside or incorporable analogue thereof,

a sugar moiety that is deoxyribose or a deoxyribose analogue; and

a cleavable blocking moiety, wherein the cleavable blocking moiety is linked to the 3′-position of the sugar moiety.

10. The method according to claim 9 wherein the unlabeled, 3′-blocked dNTP analogues are selected from the group consisting of:

(i) 3′-O-allyl-dGTP, 3′-O-allyl-dCTP, 3′-O-allyl-dATP, and 3′-O-allyl-dUTP; and

(ii) 3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dCTP, 3′-O-2-nitrobenzyl-dATP, and 3′-O-2-nitrobenzyl-dUTP.

11. The method according to claim 1 , wherein the template DNA molecules are in solution.

12. The method according to claim 1 , wherein the template DNA molecules are immobilized on a solid substrate, wherein the DNA template molecules are

bound to the solid substrate via polyethylene glycol (PEG) molecules and the solid substrate is azide-functionalized; or

immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or a biotin-streptavidin interaction.

13. The method according to claim 1 configured to use 4-channel detection, 2 channel detection, or 1 channel detection.

14. A method to determine the identity of a nucleotide residue in an extension product, comprising:

(a) combining in a sequencing reaction mixture a plurality of identical primed template DNA molecules, a DNA polymerase, and labeled, 3′-blocked dNTP analogues consisting of:

(i) a labeled, 3′-blocked dGTP analogue; and

(ii) a labeled, 3′-blocked dATP analogue; and

(iii) a labeled, 3′-blocked dCTP analogue; and

(iv) a labeled, 3′-blocked dTTP analogue and/or a labeled, 3′-blocked dUTP analogue, thereby incorporating a labeled, 3′-blocked deoxyribonucleotide triphosphate (dNTP) analogue species into at least one of said plurality of identical primed template DNA molecules and forming a labeled, blocked extension product;

(b) after step (a), removing unincorporated labeled, 3′-blocked dNTP analogues followed by adding to said sequencing reaction mixture unlabeled, 3′-blocked dNTP analogues consisting of:

(i) an unlabeled, 3′-blocked dGTP analogue; and

(ii) an unlabeled, 3′-blocked dATP analogue; and

(iii) an unlabeled, 3′-blocked dCTP analogue; and

(iv) an unlabeled, 3′-blocked dTTP analogue and/or an unlabeled, 3′-blocked dUTP analogue, incorporating an unlabeled, 3′-blocked dNTP analogue into at least one of said plurality of identical primed template DNA molecules thereby forming an unlabeled, blocked extension product; and

(c) upon addition of the unlabeled, 3′-blocked dNTP analogues during step (b), determining the identity of the labeled, 3′-blocked dNTP analogue incorporated into the labeled, blocked extension product in step (a), wherein the combined steps (a)-(c) are about 1 minute to about 10 minutes long.

15. A nucleic acid sequencing-by-synthesis method for a DNA template molecule, comprising:

(i) iterating steps (a)-(c) of claim 1 two or more times;

(ii) after each iteration, treating the distinguishable, blocked extension products and unlabeled, blocked extension products to generate extendible extension products, with the proviso that after the last iteration, treatment of the distinguishable, blocked extension products and unlabeled, blocked extension products can be omitted;

(iii) for each iteration, assigning the complement of the distinguishable, blocked dNTP analogue determined in step (c) as the identity of the nucleotide residue in the template DNA molecule for that iteration; and

(iv) assembling in order the identities of the nucleotide residues of the template DNA molecule, thereby determining by synthesis the nucleotide sequence of the template DNA molecule.