Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension

• US 20020164629A1
• Filed: 03/12/2002
• Published: 11/07/2002
• Est. Priority Date: 03/12/2001
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen, Kartalov, Emil, Braslavsky, Ido, Hebert, Benedict
• Granted Patent: US 7,297,518 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention provides methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension. Some applications of the invention utilize total internal reflection fluorescence microscopy to image polynucleotide molecules at single molecule resolution.

Claims

1. A method of analyzing sequence of a target polynucleotide, comprising:

(a) providing a primed target polynucleotide immobilized to a surface of a substrate;

wherein the target polynucleotide is attached to the surface with single molecule resolution;

(b) adding a first fluorescently labeled nucleotide to the surface of the substrate under conditions whereby the first nucleotide attaches to the primer, if a complementary nucleotide is present to serve as template in the target polynucleotide;

(c) determining presence or absence of a fluorescence signal on the surface where the target polynucleotide is immobilized, the presence of a signal indicating that the first nucleotide was incorporated into the primer, and hence the identity of the complementary base that served as a template in the target polynucleotide; and

(d) repeating steps (b)-(c) with a further fluorescently labeled nucleotide, the same or different from the first nucleotide, whereby the further nucleotide attaches to the primer or a nucleotide previously incorporated into the primer.

2. The method of claim 1, wherein step (a) comprises providing a plurality of different primed target polynucleotides immobilized to different portions of the substrate.

3. The method of claim 1, wherein steps (b)-(c) are performed at least four times with four different types of labeled nucleotides.

4. The method of claim 1, wherein steps (b)-(c) are performed until the identity of each base in the target polynucleotide has been identified.

5. The method of claim 1, further comprising an additional step of removing the signal after step (c).

6. The method of claim 1, wherein the presence or absence of a fluorescence signal is determined with total internal reflection fluorescence (TIRF) microscopy.

7. The method of claim 1, wherein the target polynucleotide is primed with a fluorescently labeled primer.

8. The method of claim 1, wherein the first and further nucleotide are labeled with the same fluorescent label.

9. The method of claim 1, wherein said the substrate is a fused silica slide.

10. The method of claim 9, wherein said surface is coated with a polyelectrolyte multilayer (PEM).

11. The method of claim 10, wherein said PEM is terminated with a polyanion.

12. The method of claim 11, wherein said polyanion bears pendant carboxylic acid groups.

13. The method of claim 12, wherein said target polynucleotide is biotinylated, and said surface is coated with streptavidin.

14. The method of claim 13, wherein said surface is coated with biotin prior to coating with streptavidin.

15. The method of claim 14, wherein said surface is coated with a polyelectrolyte multilayer (PEM) terminated with carboxylic acid groups prior to attachment of biotin.

16. The method of claim 1, wherein said removing or reducing is by photobleaching.

17. The method of claim 1, wherein the substrate is in fluid communication with a microfluidic device, wherein the first and further labeled nucleotides are added to or removed from the substrate through the microfluidic device.

18. The method of claim 17, wherein the microfluidic device comprises

(a) a flow cell comprising the substrate;

and

(b) an inlet port and an outlet port, said inlet port and outlet port being in fluid communication with said flow cell for flowing fluids into and through said flow cell.

19. The method of claim 18, wherein the substrate is a microfabricated synthesis channel.

20. The method of claim 17, furthering comprising a light source to illuminate the surface of said substrate and a detection system to detect a signal from said surface.

21. The method of claim 17, further comprising an appropriately programmed computer for recording identity of a nucleotide when said nucleotide becomes incorporated into the target polynucleotide.

22. A method of analyzing sequence of a target polynucleotide, comprising:

(a) providing a primed target polynucleotide immobilized to a surface of a substrate;

wherein the target polynucleotide is attached to the surface with single molecule resolution;

(b) adding four types of nucleotides to the surface of the substrate under conditions whereby nucleotides attach to the primer dynamically, when complementary nucleotides are present in the target polynucleotide; and

(c) monitoring in a time course of incorporation of fluorescent signals into the immobilized primer.

23. The method of claim 22, wherein monitoring of fluorescent signal incorporation into the immobilized primer is by taking images in a time course with monitored with total internal reflection fluorescence microscopy.

24. The method of claim 23, wherein the images are taken at a rate faster than the rate at which nucleotides are incorporated into the primer.

25. The method of claim 23, wherein nucleotide concentrations are low at each time point when an image is taken.

26. The method of claim 25, wherein nucleotide concentrations are alternated by fluid exchange with a microfluidic device.

27. The method of claim 22, wherein all four types of nucleotides are each labeled with a different label.

28. An apparatus for analyzing the sequence of a target polynucleotide, comprising:

(a) a flow cell comprising a substrate for immobilizing the target polynucleotide with single molecule resolution;

(b) an inlet port and an outlet port, said inlet port and outlet port being in fluid communication with said flow cell for flowing fluids into and through said flow cell;

(c) a light source for illuminating the surface of the substrate; and

(d) a detection system for detecting a signal from said surface.