Granted Patent B2

US 12,344,890 · App. 17/190,894 · Granted Jul 1, 2025

Inventors: Corey M. Dambacher (La Jolla, CA); Joseph Rokicki (San Diego, CA); Keunho Ahn (San Diego, CA); Brittany Ann Rohrman (San Diego, CA); Michael Nguyen (San Diego, CA); Kandaswamy Vijayan (San Diego, CA)

Assignee: PACIFIC BIOSCIENCES OF CALIFORNIA, INC.

C12Q1/6869C07H21/02C07H21/04C07H99/00C12Q1/6806C12Q1/6874C12N9/1241

Abstract

Claims (23)

1. A method of identifying cognate nucleotides for each of a plurality of nucleic acid features, each feature comprising a primed template nucleic acid molecule, said method comprising the steps of:

(a) preparing first stabilized ternary complexes at each of the plurality of nucleic acid features, each stabilized ternary complex comprising the primed template nucleic acid molecule at its respective nucleic acid feature, a reversible terminator nucleotide, and a polymerase;

(b) contacting the first stabilized ternary complexes from step (a) with a reagent comprising a catalytic metal ion, at least one distinguishably labeled incorporable nucleotide, and a polymerase, to produce a reaction mixture,

whereby the reversible terminator nucleotide incorporates into the primed template nucleic acid molecule to produce a blocked primed template nucleic acid molecule at each of the plurality of nucleic acid features, and

whereby there forms at each of the plurality of nucleic acid features a second stabilized ternary complex comprising the blocked primed template nucleic acid molecule and one of the distinguishably labeled incorporable nucleotides; and

(c) identifying cognate nucleotides for the primed template nucleic acid molecules at each of the plurality of nucleic acid features by detecting, in the reaction mixture, a label joined to the distinguishably labeled incorporable nucleotide of the second stabilized ternary complex.

2. The method of claim 1 , wherein the polymerase of step (b) is the same type of polymerase as in step (a).

3. The method of claim 1 , wherein the blocked primed template nucleic acid molecule of step (b) comprises a reversible terminator moiety at its 3′ terminus, and wherein the method further comprises the step of cleaving the reversible terminator moiety from the blocked primed template nucleic acid molecule to produce a 3′ terminus available for polymerization.

4. The method of claim 1 , wherein the catalytic metal ion in the second reaction mixture is selected from the group consisting of Mg 2+ ion and Mn 2+ ion.

5. The method of claim 1 , wherein the blocked primed template nucleic acid molecule of step (b) is immobilized to a solid support and wherein the solid support is contained within a flow cell.

6. The method of claim 5 , wherein each of the plurality of nucleic acid features is contained within the flow cell.

7. The method of claim 1 , wherein the label on the distinguishably labeled incorporable nucleotides is a distinguishable fluorescent label.

8. The method of claim 7 , wherein the distinguishable fluorescent label does not comprise an intercalating dye that changes fluorescence after contacting DNA.

9. The method of claim 7 , wherein the distinguishable fluorescent label is covalently attached to the incorporable nucleotide by a linker at a position on the nitrogenous base.

10. The method of claim 1 , further comprising, after step (c), the steps of:

(i) stripping the polymerase and the distinguishably labeled incorporable nucleotide of the second stabilized ternary complex from the blocked primed template nucleic acid molecule;

(ii) cleaving a reversible terminator moiety from the blocked primed template nucleic acid molecule to create a deblocked primed template nucleic acid molecule comprising a 3′ terminus available for polymerization; and

(iii) incorporating a reversible terminator into the deblocked primed template nucleic acid molecule.

11. The method of claim 1 , wherein the distinguishably labeled incorporable nucleotides in step (b) comprise at least two types of distinguishably labeled incorporable nucleotides, and wherein the detectable labels are different for each type of distinguishably labeled incorporable nucleotides.

12. The method of claim 1 , wherein the distinguishably labeled incorporable nucleotides in step (b) comprises at least three types of distinguishably labeled incorporable nucleotides, and wherein the detectable labels are different for each type of distinguishably labeled incorporable nucleotides.

13. The method of claim 1 , wherein the distinguishably labeled incorporable nucleotides in step (b) comprises four types of distinguishably labeled incorporable nucleotides, and wherein the detectable labels are different for each type of distinguishably labeled incorporable nucleotides.

14. The method of claim 1 , wherein none of the distinguishably labeled incorporable nucleotides of step (c) includes a reversible terminator moiety.

15. The method of claim 1 , wherein the reversible terminator nucleotide is unlabeled.

Assignments (3)

Continuity (6)

References Cited (184)