Method and Apparatus for Addressable Flow Cells in Single Molecule Sequencing

• US 20110311963A1
• Filed: 03/16/2011
• Published: 12/22/2011
• Est. Priority Date: 03/16/2010
• Status: Abandoned Application

• Current Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: BEECHEM, JOSEPH, LAFFERTY, W. MICHAEL, SUN, HONGYE, METZKER, MICHAEL
• Application Number:
  US13/049,660
• Publication Number:
  US 20110311963A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides

Abstract

A method of sequencing a plurality of template nucleotide sequences includes immobilizing the plurality of template nucleotide sequences on a substrate. A first subset of the plurality of template nucleotide sequences is immobilized in a first field of view and a second subset of the plurality of template nucleotide sequences is immobilized in a second field of view. The first and second subsets are hybridized to a caged primer. The caged primer includes a caging group. The method further includes lysing the caging group from the caged primer in the first field of view and observing the first field of view to detect sequencing of the first subset of the plurality of template nucleotide sequences.

Claims

1. A method of sequencing a plurality of template nucleotide sequences, the method comprising:

immobilizing the plurality of template nucleotide sequences on a substrate, a first subset of the plurality of template nucleotide sequences immobilized in a first field of view and a second subset of the plurality of template nucleotide sequences immobilized in a second field of view, the first and second subsets hybridized to a caged primer, the caged primer including a caging group;

lysing the caging group from the caged primer in the first field of view; and

observing the first field of view to detect sequencing of the first subset of the plurality of template nucleotide sequences.

2. The method of claim 1, wherein lysing the caging group includes photolysing the caging group from the caged primer.

3. The method of claim 2, wherein photolysing includes directing electromagnetic radiation having a wavelength in a range of 100 nm to 1000 nm.

4. The method of claim 1, further comprising lysing the caging group from the caged primers in the second field of view and observing the second field of view.

5. The method of claim 1, wherein a third subset of the plurality of template nucleotide sequences is hybridized to a second caged primer having a second caging group different from the caging group, the method further comprising lysing the second caged group from the second caged primer.

6. The method of claim 5, wherein lysing the second caged group includes photolysing the second caged group.

7. The method of claim 6, wherein photolysing the second caged group includes photolysing using a wavelength different from a wavelength used during lysing the caging group.

8. The method of claim 1, wherein observing the first field of view includes optically detecting incorporation of nucleotides.

10. The method of claim 1, wherein the caging group is selected from the group consisting of nitrobenzyl, α

-carboxy-2-nitrobenzyl (CNB), 1-(2-nitrophenyl)ethyl (NPE), 4,5-dimethoxy-2-nitrobenzyl (DMNB), 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE), 5-carboxymethoxy-2-nitrobenzyl (CMNB) caging groups, and any combination thereof.

11. The method of claim 10, wherein the caging group is a nitrobenzyl caging group.

14. The method of claim 10, wherein the caging group is a dimethoxy caging group.

9. (canceled)

12. (canceled)

13. (canceled)

15-16. -16. (canceled)

17. A method of sequencing a template nucleotide sequences, the method comprising:

applying a caged primer to the template nucleotide sequences, the caged primer including an oligonucleotide primer and a caging group coupled to the oligonucleotide primer; and

lysing the caged group from the caged primer.

18. The method of claim 17, wherein the caging group is coupled to a terminal end of the oligonucleotide primer.

19. The method of claim 18, wherein the terminal end is the 3′

terminal end.

20. The method of claim 17, further comprising applying polymerase and a nucleotide to the template nucleotide sequences and observing to detect incorporation of the nucleotide.

21. The method of claim 20, wherein the nucleotide is coupled to a fluorescent dye, the fluorescent dye fluorescing in response to incorporation, and wherein observing includes optically detecting.

22. The method of claim 17, wherein lysing the caged group includes photolysing the caged group.

24. The method of claim 17, wherein the caged primer includes nitrobenzyl functionality.

32. The method of claim 17, further comprising immobilizing the template nucleotide sequences on a substrate.

23. (canceled)

25-31. -31. (canceled)

33-38. -38. (canceled)

39. A kit to sequence template nucleotide sequences, the kit comprising:

a substrate to immobilize template nucleotide sequences, the substrate sized to include a plurality of fields of view; and

a caged primer including an oligonucleotide primer and a caging group coupled to a terminal end of the oligonucleotide primer.

40-65. -65. (canceled)