Apparatus and methods for kinetic analysis and determination of nucleic acid sequences

Patent metadata

Field	Value
Publication number	US-9279154-B2
Application number	US-201213722979-A
Country	US
Kind code	B2
Filing date	Dec 20, 2012
Priority date	Dec 21, 2011
Publication date	Mar 8, 2016
Grant date	Mar 8, 2016

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.

First claim

What is claimed is:

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 to cause binding of the polymerase molecules to the nucleic acid molecules,
wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features at a density of at least 100 features/cm 2 , and
wherein the mixing comprises stopped flow delivery of the polymerase molecules to the nucleic acid features;
(b) monitoring the 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 determining 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, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules based on the pre-steady state kinetic rate profiles.

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 nucleotide molecules to the nucleic acid features.

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

11. The method of claim 10 , 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.

12. The method of claim 11 , wherein the method is performed sequentially for four different nucleotide species that comprise reversible blocking moieties.

13. The method of claim 12 , wherein the method is repeated several times to determine the nucleotide sequences for the different nucleic acid molecules.

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

15. 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.

16. 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.

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

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

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

20. The method of claim 1 , wherein each feature of the array comprises only a single nucleic acid molecule.

21. The method of claim 1 , wherein each feature of the array comprises a population of molecules of the same nucleic acid species.

22. The method of claim 1 , wherein the stopped flow delivery has a dead time of less than 2 milliseconds.

23. The method of claim 1 , wherein the mixing is achieved at a flow rate of at least 20 ml/min.

Assignees

• Illumina Inc

Inventors

• Previte Michael
• He Molly
• Pantoja Rigo
• Chen Cheng-Yao
• Zhou Chunhong

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2561/12

  Fluorescence lifetime measurement · CPC title

• C12Q2537/149

  Sequential reactions · CPC title

• C12Q2565/518

  characterised by the immobilisation of the nucleic acid sample or target · CPC title