Scanning system and method for imaging and sequencing

• US 20090250615A1
• Filed: 04/06/2009
• Published: 10/08/2009
• 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.
• Granted Patent: US 8,834,797 B2
• Time in Patent Office: Days
• US Class Current: 250/362
• 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 wherein emissions from locations in a flow cell are detected. In some embodiments, the system can comprise 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 system for detecting emissions from a plurality of fixed locations on a substrate, the system comprising:

a first irradiation source configured to irradiate the substrate and disposed for irradiating a first group of fixed locations of the plurality of fixed locations;

a second irradiation source configured to irradiate a second group of fixed locations of the plurality of fixed locations, the second irradiation source being fixed with respect to the first irradiation source such that the second group of fixed locations is spaced from and excludes the first group of fixed locations on the substrate;

a scanning detector array positioned with respect to the first irradiation source and the second irradiation source so as to collect emission from the first group of fixed locations without collecting emission from the second group of fixed locations; and

a translation stage configured to move the first irradiation source, the second irradiation source, and the scanning detector array, together.

2. The system of claim 1, wherein at each location a polynucleotide is disposed and at least some of the polynucleotides are labeled with respective markers that fluoresce when exposed to excitation light.

3. The system of claim 1, further comprising a substrate, wherein the substrate is positioned with respect to the first irradiation source so as to be irradiated by the first irradiation source.

4. The system of claim 3, wherein the substrate comprises a flow cell and a plurality of labeled polynucleotides disposed in the flow cell.

5. The system of claim 1, wherein the first irradiation source comprises at least one laser source, a holographic beam shaper, and a trombone prism.

6. The system of claim 1, wherein the scanning detector array comprises a two-dimensional charge-coupled device.

7. The system of claim 1, wherein the scanning detector array comprises a point detector.

8. The system of claim 1, wherein the scanning detector array comprises a time-delay integration image detector.

9. A system for detecting fluorescently labeled polynucleotides, comprising:

a substrate comprising a first end, and a second end, one or more channels formed therein, and a plurality of labeled polynucleotide beads immobilized in the one or more channels, each of the labeled polynucleotide beads having a diameter and being labeled with a respective marker that is responsive to radiation and that emits emission light indicative of the presence of at least one nucleotide;

at least one irradiation source for emitting radiation and disposed for irradiating the plurality of labeled polynucleotide beads to thereby excite the markers responsive to the radiation and to cause the markers to emit emission light;

modulating optics comprising at least one of a cylindrical lens and a holographic beam shaper, the modulating optics configured to shape radiation emitted from the at least one irradiation source onto a portion of the labeled polynucleotide beads;

a scanning detector array positioned with respect to the plurality of labeled polynucleotide beads, the scanning detector array configured to collect the emission light produced by the markers and to produce charges corresponding to the emission light, the detector array having an output; and

a time-delay integration (TDI) system that accumulates charges corresponding to the emission light, within the detector array, and reads accumulated charges at the output of the scanning detector array.

10. The system of claim 9, further comprising second modulating optics disposed between the at least one irradiation source and the scanning detector array, the second modulating optics comprising at least one of a cylindrical lens, a trombone prism, and a holographic beam shaper.

11. The system of claim 9, wherein the TDI system is configured to accumulate charge by moving, relative to one another, the at least one irradiation source and the plurality of labeled polynucleotide beads, without moving the at least one irradiation source and the scanning detector array relative to one another.

12. The system of claim 9, wherein the one or more channels comprises from two to 10 channels, and the scanning detector array is configured to collect emission light produced by at least two channels sequentially.

13. A method for detecting a sequence of nucleotides of a polynucleotide, the method comprising:

causing a first reaction in a flow cell, the flow cell comprising a plurality of fixed locations and a plurality of polynucleotides immobilized at the plurality of fixed locations, the first reaction involving at least some of the plurality of polynucleotides;

generating a first beam of radiation with an excitation source;

directing the first beam at the flow cell;

scanning the flow cell with a scanning detector array, to detect radiation emitted from the plurality of fixed locations;

causing a second reaction in the flow cell, the second reaction involving at least some of the polynucleotides;

irradiating the flow cell with a second beam of radiation subsequent to the second reaction;

scanning the flow cell with the scanning detector array subsequent to the second reaction, to detect radiation emitted from the plurality of fixed locations; and

using information ascertained from the scanning steps to determine a sequence of nucleotides of one or more of the plurality of nucleotides.

14. The method of claim 13, wherein the first reaction comprises a ligation reaction.

15. The method of claim 13, further comprising the step of photocleaving protective groups from one or more of the plurality of polynucleotides after one or more of the scanning steps.

16. The method of claim 13, wherein the flow cell comprises a plurality of beads therein, the plurality of polynucleotides are immobilized on the plurality of beads, and the plurality of fixed locations are locations occupied by the plurality of beads.

17. The method of claim 13, wherein the plurality of polynucleotides comprises a plurality of single molecule polynucleotides each immobilized at a respective one of the fixed locations.

18. The method of claim 13, further comprising shaping the first beam, the second beam, and the third beam with modulating optics disposed between the excitation source and the flow cell, wherein the modulating optics comprises at least one of a cylindrical lens, a holographic beam shaper, and a trombone prism.

19. The method of claim 13, wherein the plurality of polynucleotides are immobilized on a plurality of beads, the flow cell comprises a plurality of grooves, and the beads are disposed in the plurality of grooves.

20. The method of claim 13, wherein the plurality of polynucleotides comprise at least some polynucleotides bearing protective groups, and the method further comprises photocleaving the protective groups with a photocleavage beam source that differs from the excitation source.