APPARATUS AND METHODS FOR KINETIC ANALYSIS AND DETERMINATION OF NUCLEIC ACID SEQUENCES

• US 20130165328A1
• Filed: 12/20/2012
• Published: 06/27/2013
• Est. Priority Date: 12/21/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Previte, Michael, He, Molly, Pantoja, Rigo, Chen, Cheng-Yao, Zhou, Chunhong
• Granted Patent: US 9,279,154 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

A method of distinguishing nucleotide sequences for different nucleic acid molecules including the steps of (a) mixing a plurality of different nucleic acid molecules with polymerase molecules and nucleotide molecules, wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features; (b) determining a transient state of the polymerase molecules at the nucleic acid features; and (c) identifying a subset of nucleic acid features that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

Claims

1. A method of distinguishing nucleotide sequences for different nucleic acid molecules, comprising

(a) mixing a plurality of different nucleic acid molecules with polymerase molecules and nucleotide molecules,

wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features;

(b) monitoring binding of the polymerase molecules to the nucleic acid features at several points during a pre-equilibrium time period, thereby determining a transient state of the polymerase molecules at the nucleic acid features; and

(c) identifying nucleic acid features of the array that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

2. The method of claim 1, wherein (b) comprises monitoring pre-steady state kinetic rate profiles of the binding of the polymerase molecules to the nucleic acid features.

3. The method of claim 2, wherein the monitoring of the pre-steady state kinetic rate profiles occurs before, during, and after correct incorporation of the nucleotide molecules into the nucleic acid features.

4. The method of claim 2, wherein (c) comprises identifying the nucleic acid features of the array that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features, whereby the kinetic rate profiles determine nucleotide molecules that are correctly incorporated into the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

5. The method of claim 1, further comprising

(d) removing the polymerase molecules from the nucleic acid features, thereby providing restored features;

(e) mixing the restored features with polymerase molecules and a second species of nucleotide molecules, wherein the second species of nucleotide molecules is different from the species of nucleotide molecules in (a); and

(f) repeating (b) and (c) for the restored features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

6. The method of claim 5, wherein the method is performed sequentially for four different nucleotide species.

7. The method of claim 6, wherein the method is repeated several times to determine the nucleotide sequences for the different nucleic acid molecules.

8. The method of claim 5, wherein (d) further comprises removing the nucleotide molecules from the nucleic acid features.

9. The method of claim 1, wherein the mixing comprises stopped flow delivery of the polymerase molecules to the nucleic acid features.

10. The method of claim 1, wherein the mixing comprises stopped flow delivery of the nucleotide molecules to the nucleic acid features.

11. The method of claim 1, wherein the one or more of the nucleotide molecules comprise reversible blocking moieties.

12. The method of claim 11, further comprising

(d) removing the polymerase molecules from the nucleic acid features, and removing the blocking moieties at the nucleic acid features that correctly incorporate the nucleotide molecules, thereby providing restored features;

(e) mixing the restored features with polymerase molecules and a second species of nucleotide molecules, wherein the second species of nucleotide molecules is different from the species of nucleotide molecules in (a); and

(f) repeating (b) and (c) for the restored features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

13. The method of claim 12, wherein the method is performed sequentially for four different nucleotide species that comprise reversible blocking moieties.

14. The method of claim 13, wherein the method is repeated several times to determine the nucleotide sequences for the different nucleic acid molecules.

15. The method of claim 1, wherein the transient state is determined from the time duration for the binding to reach equilibrium.

16. The method of claim 1, wherein the determining of the transient state comprises determining binding rate constants for the binding of the polymerase molecules to the nucleic acid features.

17. The method of claim 1, wherein the determining of the transient state comprises determining catalytic rate constants for incorporation of the nucleotide molecules into the nucleic acid features.

18. The method of claim 1, wherein the polymerase molecules comprise labels and the monitoring in (b) comprises detecting the labels.

19. The method of claim 1, wherein the nucleotide molecules comprise labels and the monitoring in (b) comprises detecting the labels.

20. A system for determining sequences of nucleic acids from pre-equilibrium kinetic measurements of extension reactions for the nucleic acids, the system comprising

(a) an array comprising nucleic acid features having different nucleotide sequences;

(b) a fluidic apparatus configured to deliver sequencing reagents to the array, wherein the sequencing reagents comprise polymerase molecules and nucleotide molecules for the nucleic acid extensions reactions;

(c) a detection apparatus configured to obtain the kinetic measurements from the array at a resolution that distinguishes individual nucleic acid features of the array;

(d) a control module comprising instructions for

(i) directing the fluidic apparatus to deliver the sequencing reagents to the array at an initiation time point, and

(ii) directing the detection apparatus to obtain the kinetic measurements during the pre-equilibrium time period relative to the initiation time point; and

(e) an analysis module comprising instructions for

(i) processing the kinetic measurements to determine binding of the polymerase molecules to the nucleic acid features at several points during the pre-equilibrium time period, thereby determining a transient state of the polymerase molecules at the nucleic acid features, and

(ii) identifying nucleic acid features that correctly incorporate the nucleotide molecules based on the transient state of the polymerase molecules at the nucleic acid features.