Methods and apparatus for analyzing polynucleotide sequences by asynchronous base extension

• US 7,297,518 B2
• Filed: 03/12/2002
• Issued: 11/20/2007
• Est. Priority Date: 03/12/2001
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen, Kartalov, Emil, Braslavsky, Ido, Hebert, Benedict
• Primary Examiner(s): Lu; Frank W.
• Application Number:
  US10/099,459
• Publication Number:
  US 20020164629A1
• Time in Patent Office: 2,079 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/287.1, 435/287.2, 435/288.4, 435/288.7, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• 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 comprising a substantially complementary fluorescently labeled primer and a target polynucleotide, the primed target polynucleotide being immobilized to a substrate comprising a polyelectrolyte coated multilayer surface, wherein the target polynucleotide is randomly attached to the polyelectrolyte multilayer coated surface with single molecule resolution;

(b) adding a first fluorescently labeled nucleotide to the surface of the substrate under conditions whereby the first nucleotide is added to the primer;

(c) determining whether a fluorescence signal from the fluorescently labeled nucleotide is present in at least one molecule of the primed target polynucleotide on the surface of the substrate, wherein the presence of the signal from the fluorescently labeled nucleotide indicates that the fluorescent nucleotide is added to the primer;

(d) removing the fluorescent signal from the fluorescently labeled nucleotide; and

(e) repeating steps (b)-(d) with a further fluorescently labeled nucleotide, thereby analyzing the sequence of the target polynucleotide, wherein if the primer label and the nucleotide label are the same, detecting and then bleaching the fluorescent signal from the primer label prior to adding the first fluorescently labeled nucleotide.

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)-(d) are performed with at least four different types of fluorescently labeled nucleotides.

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

5. The method of claim 1, wherein presence or absence of fluorescence signal from the fluorescently labeled nucleotide is determined with total internal reflection fluorescence (TIRF) microscopy.

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

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

8. The method of claim 1, wherein said polyelectrolyte multilayer is terminated with a polyanion.

9. The method of claim 8, wherein said polyanion bears pendant carboxylic acid groups.

10. The method of claim 9, wherein said target polynucleotide is biotinylated, and said surface is coated with streptavidin.

11. The method of claim 10, wherein said surface is coated with biotin prior to coating with streptavidin.

12. The method of claim 1, wherein said removing is by photobleaching.

13. 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.

14. The method of claim 13, 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.

15. The method of claim 14, wherein the substrate is a microfabricated synthesis channel.

16. The method of claim 13, further comprising a light source to illuminate the surface of said substrate and a detection system to detect a signal from said surface.

17. The method of claim 13, further comprising an appropriately programmed computer for recording an identity of a nucleotide when said nucleotide is added to said primer or to a nucleotide previously added to the primer.

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

(a) providing a primed target polynucleotide comprising a substantially complementary fluorescently labeled primer and a target polynucleotide, the primed target polynucleotide being immobilized to a substrate comprising a coated surface, wherein the target polynucleotide is randomly attached to the surface with single molecule resolution;

(b) adding four types of nucleotides to the surface of the substrate under conditions whereby nucleotides are added to the primer dynamically, at least one type of nucleotide being fluorescently labeled; and

(c) monitoring a time course of fluorescent signal from addition of at least one fluorescent nucleotide to the primer, thereby analyzing the sequence of the target polynucleotide, wherein if the primer label and the nucleotide label are the same, detecting and then bleaching the signal from the primer label prior to adding the four types of nucleotides.

19. The method of claim 18, wherein said monitoring step comprises taking images in the time course with total internal reflection fluorescence microscopy.

20. The method of claim 19, wherein the images are taken at a rate faster than the rate at which the fluorescently labeled nucleotide is added to the primer.

21. The method of claim 19, wherein fluorescence signal from addition of the fluorescently labeled nucleotide is detectable in the presence of fluorescent signal from unincorporated fluorescently labeled nucleotides.

22. The method of claim 18, wherein concentrations of the nucleotides are alternated by fluid exchange with a microfluidic device.

23. The method of claim 18, wherein the four types of nucleotides are each labeled with a different label.

24. The method of claim 18, wherein the surface is coated with a polyelectrolyte multilayer.