Scanning system and method for imaging and sequencing

• US 8,834,797 B2
• Filed: 04/06/2009
• Issued: 09/16/2014
• Est. Priority Date: 04/04/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Oldham, Mark F., Nordman, Eric S., Sun, Hongye, Boege, Steven J.
• Primary Examiner(s): HIXSON, CHRISTOPHER
• Application Number:
  US12/384,516
• Publication Number:
  US 20090250615A1
• Time in Patent Office: 1,989 Days
• Field of Search: 422/50, 422/425, 422/68.1, 422/82.05, 422/82.07, 422/82.08, 422/501, 422/502, 422/503, 436/94, 436/164, 436/172, 435/287.1, 435/287.2, 435/287.9, 506/33, 506/34, 506/35, 506/37, 506/39, 356/444
• US Class Current: 422/82.08
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

A scanning detection system is provided in which emissions from locations in a flow cell are detected. In some embodiments, the system can include an excitation source, a photocleavage source, and modulating optics configured to cause an excitation beam generated by the excitation source to irradiate a first group of the fixed locations and to cause a photocleavage beam generated by the photocleavage source to irradiate a second group of the fixed locations, which is separate and apart from the first group of fixed locations. Methods of detecting sequencing reactions using such a system are also provided.

Claims

1. A polynucleotide sequencing system, comprising:

a flow cell having at least one channel, a plurality of polynucleotides disposed in the at least one channel, at least a portion of the plurality of polynucleotides comprising a detectable label;

a first irradiation source configured to generate a first excitation beam which irradiates a first group of polynucleotides of the plurality of polynucleotides thereby exciting at least one detectable label of at least one polynucleotide of the first group of polynucleotides such that the at least one detectable label of the at least one polynucleotide of the first group of polynucleotides emits an emission signal;

a second irradiation source configured to generate a second excitation beam which irradiates a second group of polynucleotides of the plurality of polynucleotides thereby exciting at least one detectable label of at least one polynucleotide of the second group of polynucleotides such that the at least one detectable label of the at least one polynucleotide of the second group of polynucleotides emits an emission signal, the second group of polynucleotides being different from the first group of polynucleotides, the first and second excitation beams simultaneously irradiating the first and second groups of polynucleotides, respectively, wherein the first excitation beam does not substantially overlap the second excitation beam;

a detection system having at least a first detector configured to detect the emission signal from the at least one detectable label of the at least one polynucleotide of the first group of polynucleotides and having at least a second detector configured to detect the emission signal from the at least one detectable label of the at least one polynucleotide of the second group of polynucleotides, the first and second detectors configured to detect distinct wavelengths or wavelength ranges relative to one another corresponding to distinct nucleotides; and

a computer in operative communication with the detection system and configured to collect and process the detectable emissions from the first and second groups of polynucleotides.

2. The system of claim 1, wherein the first irradiation source comprises at least one laser source.

3. The system of claim 1, wherein at least one detector of the detection system comprises a charge-coupled device.

4. The system of claim 1, wherein the detection system comprises a point detector.

5. The system of claim 1, wherein at least one detector of the detection system comprises a time-delay integration image detector.

6. The system of claim 1, wherein the first group of polynucleotides partially overlaps the second group of polynucleotides.

7. The system of claim 1, wherein the first group of polynucleotides excludes the second group of polynucleotides.

8. The system of claim 1, wherein the plurality of polynucleotides and the first and second excitation beams are stationary relative to one another as the detection system detects emissions.

9. The system of claim 1, wherein the plurality of polynucleotides and the first and second excitation beams are moving relative to one another as the detection system detects emissions.

10. The system of claim 1, wherein the emission signal from the at least one detectable label of the at least one polynucleotide of the first group of polynucleotides is indicative of a nature of the at least one polynucleotide of the first group of polynucleotides, and the emission signal from the at least one detectable label of the at least one polynucleotide of the second group of polynucleotides is indicative of a nature of the at least one polynucleotide of the second group of polynucleotides.

11. The system of claim 1, wherein the emission signal from the at least one detectable label of the at least one polynucleotide of the first group of polynucleotides is indicative of a presence of a nucleotide of the at least one polynucleotide.

12. The system of claim 1, wherein the emission signal from the at least one detectable label of the at least one polynucleotide of the first group of polynucleotides is indicative of an identity of a nucleotide of the at least one polynucleotide.

13. The system of claim 1, wherein each detectable label is one of at least 4 distinct, fluorescent labels, each of the 4 distinct, fluorescent labels being indicative of a distinct result of a sequencing reaction.

14. The system of claim 13, wherein each of the 4 distinct, fluorescent labels are each indicative of the presence or nature of a distinct nucleotide.

19. The system of claim 1, wherein the detection system comprises 4 detectors, each detector being a camera configured to detect an emission signal of a distinct wavelength or wavelength range, each wavelength or wavelength range corresponding to a distinct nucleotide.

20. The system of claim 1, further comprising a stage configured to support the flow cell, the stage further configured to move the flowcell relative to the first and second excitation beams.

21. The system of claim 1, wherein the polynucleotides are fixed relative to the channel.

22. The system of claim 21, wherein the at least one channel is formed at least in part between a top flow cell wall and a bottom flow cell substrate, at least a portion of the plurality of polynucleotides being disposed on an underside of the top flow cell wall, a portion of the polynucleotides disposed on the underside of the top flow cell wall comprising a detectable label.

23. The system of claim 21, wherein the system is configured to move the flow cell relative to the detection system such that the detection system detects emissions in a first direction along the flow cell, the system further configured to move the flow cell a distance in an axis orthogonal to a length of the at least one channel, and further configured to subsequently move the flow cell relative to the detection system such that the detection system detects emissions in a second direction along the flow cell.

24. The system of claim 1, further comprising more than one flow cell.

25. The system of claim 1, wherein the first excitation beam includes radiation of a first wavelength or wavelength range, and the second excitation beam includes radiation of a second wavelength or wavelength range, the first wavelength or wavelength range being different from the second wavelength or wavelength range.

26. The system of claim 1, further comprising modulating optics disposed between at least one of the first and second irradiation sources and the flow cell.

28. The system of claim 23, wherein the first direction is the same as the second direction.

29. The system of claim 23, wherein the first direction is opposite of the second direction.

30. The system of claim 1, wherein at least one detector of the detection system comprises a complimentary metal-oxide-semiconductor (CMOS) device.

15. A system for detecting emissions, comprising:

a flow cell comprising one or more channels, a plurality of polynucleotides in at least one of the channels;

a first irradiation source configured to generate a first radiation beam which irradiates a first group of polynucleotides of the plurality of polynucleotides, at least one polynucleotide of the first group of polynucleotides configured to emit a detectable emission in response to the first radiation beam;

a second irradiation source configured to generate a second radiation beam which irradiates a second group of polynucleotides of the plurality of polynucleotides, at least one polynucleotide of the second group of polynucleotides configured to emit a detectable emission in response to the second radiation beam, the second group of polynucleotides being different from the first group of polynucleotides, the first and second radiation beams simultaneously irradiating the first and second groups of polynucleotides, respectively, wherein the first radiation beam does not substantially overlap the second radiation beam;

a detection system having at least four time-delay integration detectors, the system configured to detect at least the detectable emission produced by the at least one polynucleotide of the first group of polynucleotides and the detectable emission produced by the at least one polynucleotide of the second group of polynucleotides, the system configured such that at least one of the flow cell, the first group of polynucleotides, the second group of polynucleotides, and the plurality of polynucleotides move relative to the at least four time-delay integration detectors during detection of the emissions by the detection system, each of the at least four time-delay integration detectors configured to detect a distinct wavelength or wavelength range relative to the other time-delay integration detectors, the distinct wavelengths or wavelength ranges corresponding to distinct nucleotides; and

a computer in operative communication with the detection system and configured to collect and process the detectable emissions from the first and second groups of polynucleotides.

16. The system of claim 15, wherein a portion of the plurality of polynucleotides are labeled with a fluorescent label or fluorescent marker configured to emit an emission of an emission wavelength or wavelength range in response to being irradiated by an excitation wavelength or wavelength range.

17. The system of claim 16, wherein the first radiation beam includes a first excitation wavelength or wavelength range, and the second radiation beam includes a second excitation wavelength or wavelength range, the first excitation wavelength or wavelength range being different from the second excitation wavelength or wavelength range.

18. The system of claim 15, wherein the one or more channels comprises from two to 10 channels, and the detection system is configured to detect emissions from polynucleotides within at least two channels sequentially.

27. The system of claim 15, wherein the plurality of polynucleotides are fixed relative to the channel.

31. The system of claim 15, wherein at least one detector of the detection system comprises a complimentary metal-oxide-semiconductor (CMOS) device.