SYSTEMS AND DEVICES FOR SEQUENCE BY SYNTHESIS ANALYSIS

• US 20100111768A1
• Filed: 03/30/2007
• Published: 05/06/2010
• Est. Priority Date: 03/31/2006
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Incorporated (Illumina Incorporated)
• Inventors: Bridgham, John, Lundberg, Peter, Barnes, Colin, Pratt, Mark, Obradovic, Bojan, Etchin, Sergey, Banerjee, Saibal, Maisinger, Klaus, Swerdlow, Harold, Sankar, Subra, Ho, Jonny, Rodighiero, Chiara, Vermaas, Eric, Benson, Kevin, Schroth, Gary, Lee, Xavier, Rasolonjatovo, Isabelle, Sizto, Ning, Buermann, Dale, Reed, Mark, Bryant, Jason
• Granted Patent: US 8,241,573 B2
• Time in Patent Office: Days
• US Class Current: 422/82.80
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/60 Detection means characterised by use of a special device
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging

Abstract

The present invention comprises systems and devices for sequencing of nucleic acid, such as short DNA sequences from clonally amplified single-molecule arrays.

Claims

1. A system for sequencing one or more polynucleotide, the system comprising:

a) a solid substrate comprising one or more polynucleotides attached thereto;

b) a fluid direction system for controllably moving one or more fluorescently labeled reagents into contact with the polynucleotides;

c) a temperature control system for regulating the temperature of at least one of the substrate and the reagents;

d) an illumination system for exciting the fluorescent label via total internal reflection (TIR);

e) a detector component proximal to the substrate for detecting fluorescence produced from excitation of the fluorescent label by the TIR system;

f) a computer system operably coupled to the detector wherein the computer comprises an instruction set for acquiring fluorescence images from the detector.

2. The system of claim 1, wherein the substrate can be moved distal to the detector in order for the temperature control system to regulate the temperature of the substrate.

3. The system of claim 1, wherein the solid substrate comprises a flowcell, which flowcell comprises one or more fluidic channels in which the polynucleotides are attached.

4. The system of claim 1, wherein the substrate comprises glass, silicon, or plastic.

5. The system according to claim 1, wherein said solid substrate is an array of beads.

6. The system of claim 1, wherein the reagents comprise components to extend a second sequence complementary to the one or more polynucleotides.

7. The system according to claim 6, wherein said reagents are fluorescently labeled nucleoside triphosphates.

8. The system according to claim 6, wherein said reagents are fluorescently labeled oligonucleotides.

9. The system of claim 1, wherein the illumination system comprises at least one excitation laser coupled through an optical fiber.

10. The system according to claim 9, wherein said fiber is physically deformed to obtain a uniform illumination footprint.

11. The system according to claim 10, wherein said physical deformation is squeezing.

12. The system according to claim 10, wherein said physical deformation is vibrating.

13. The system of claim 1, wherein the detector comprises a CCD camera.

14. The system of claim 1, wherein said detector comprises two CCD cameras.

15. The system of claim 1, wherein said detector comprises four CCD cameras.

16. The system of claim 1, wherein the detector further comprises one or more optic filters appropriate to the fluorescence emission of the fluorescently labeled reagent and to one or more wavelength of light from the laser.

17. The system of claim 1, when the detector component comprises an autofocus mechanism.

18. The system of claim 1 comprising two fluidics stations for operating on two flowcells simultaneously.

19. The system of claim 1, wherein the illumination system comprises two excitation lasers coupled through a fiberoptic device, wherein such lasers illuminate at least part of the same area.

20. The system of claim 16, wherein the reagents include four different reagents having respective fluorescent labels, the detector further comprises four optic filters appropriate to the fluorescence emission of the four fluorescently labeled reagents and to the wavelength of light from the lasers.

21. The system of claim 19, wherein said lasers emit at a wavelength of 532 nm and 660 nm.

22. The system of claim 3, wherein the flowcell comprises a channel layer and a cover layer that is overlaid upon the channel layer, the channel layer including the fluidic channel, the cover layer having a through-hole that provides access to the fluidic channel.

23. The system of claim 22, wherein the channel layer comprises photosensitive glass.

24. The system of claim 3, wherein the fluidic channel has one or more inlet ports at a first end of the channel and one or more outlet ports at a second end of the channel, the inlet and outlet ports being located with respect to each other to facilitate maintaining a uniform flow of a liquid through the fluidic channel.

25. The system of claim 9, wherein said fiber includes a core having a substantially rectangular cross-sectional shape.

26. The system of claim 1, wherein the illumination system is configured to provide an autofocus spot that is detected by the detector, the computer system using the autofocus spot to focus the detector.

27. The system of claim 1, wherein the polynucleotides comprise DNA clusters and the reagents comprise bases having the fluorescent labels, each DNA cluster including a plurality of single-stranded DNA fragments having a common sequence;

and

wherein the fluid direction system, the temperature control system, the illumination system, the detector component, and the computer system cooperate with one another to perform a DNA sequencing cycle including the steps of;

(i) incorporating at least one base onto the DNA fragments of the DNA clusters;

(ii) illuminating the DNA clusters to excite the fluorescent labels of the bases;

(iii) capturing an image of emitted fluorescence from the DNA clusters; and

(iii) identifying the base incorporated onto the DNA fragments of the DNA clusters based upon the emitted fluorescence from the DNA clusters;

(iv) removing the fluorescent labels from the bases.

28. The system of claim 27, wherein the base is one of four types of bases, the bases of each type having a unique fluorescent label.

29. The system of claim 27, wherein the computer system is configured to repeat steps (i)-(iv) to determine the sequences of the DNA fragments.

30. The system of claim 1, wherein the reagents include four different bases in which each base has a different fluorescent label, the illumination system including first and second lasers, each of the first and second lasers exciting two of the four fluorescent labels so that the four fluorescent labels are excited by the first and second lasers.

31. The system of claim 30 further comprising four different optical filters for selecting the fluorescence emission wherein two of the four filters have a narrower bandwidth than the other two of the four filters.

32. The system of claim 3 wherein the fluid direction system comprises a pump placed after the flowcell to pull reagents through the flowcell.

33. The system of claim 27 wherein the detector is a CCD camera and a density of DNA clusters recorded on the CCD camera is about 5,000 to 50,000 clusters per million pixels.