Sample preparation method and apparatus for nucleic acid sequencing

• US 20070128610A1
• Filed: 12/02/2005
• Published: 06/07/2007
• Est. Priority Date: 12/02/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Buzby, Philip
• Application Number:
  US11/292,528
• Publication Number:
  US 20070128610A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Abstract

The invention provides methods and apparatus for preparation and sequencing of nucleic acids.

Claims

1. A method for sequencing a nucleic acid, the method comprising the steps of:

a) introducing one or more cells into a flow cell;

b) treating said cells to cause nucleic acids to be released;

c) immobilizing released nucleic acids on a surface of the flow cell; and

d) conducting a sequencing reaction using said immobilized nucleic acids as templates.

2. The method of claim 1, wherein a single cell is introduced into the flow cell.

3. The method of claim 1, further comprising the step of fragmenting said released nucleic acids.

4. The method of claim 1, wherein the cells are lysed and the released nucleic acids are immobilized in separate regions of the flow cell.

5. The method of claim 1, wherein said surface is coated with an epoxide or epoxide derivative.

6. The method of claim 5, wherein said epoxide is derivatized with a member of a binding pair.

7. The method of claim 6, wherein said member of a binding pair is selected from the group consisting of antibodies, antigens, receptors, and ligands.

8. The method of claim 3, wherein the released nucleic acids are fragmented by a method selected from the group consisting of sonication, and enzymatic digestion.

9. The method of claim 1, wherein the immobilized nucleic acids are exposed to primers under conditions suitable for forming a template/primer duplex.

10. The method of claim 9, wherein the primers comprise a homopolymeric nucleotide sequence.

11. The method of claim 9, wherein step d) comprises the steps of:

e) introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition to said primer;

f) removing unincorporated nucleotide; and

g) identifying nucleotide species incorporated into said primer.

12. The method of claim 1, wherein the cells are isolated from a biological sample prior to introducing the cells into the flow cell.

13. The method of claim 1, wherein the released nucleic acids are modified with a member of a binding pair and the surface comprises another member of said binding pair, for immobilizing the nucleic acids.

14. A method for sequencing a nucleic acid, the method comprising the steps of:

exposing cells to a flow cell, the flow cell comprising an inlet and an outlet;

extracting nucleic acids from said cells;

attaching said nucleic acids to a surface associated with said flow cell, such that at least a portion of said nucleic acids are individually optically resolvable;

hybridizing a primer to said nucleic acids to form template/primer duplexes;

exposing said duplexes to optically-labeled nucleotides and polymerase under conditions that allow template-dependent nucleotide addition to said primer; and

identifying nucleotides added to said primer.

15. The method of claim 14, wherein about 1000 cells are exposed to the flow cell.

16. The method of claim 14, further comprising the step of fragmenting said nucleic acids prior to attachment to said surface.

17. The method of claim 14, further comprising the step of adding a predetermined number of nucleotides to the 3′

end of said nucleic acids.

18. The method of claim 17, wherein said primer is complementary to said predetermined number of nucleotides.

19. The method of claim 14, wherein said nucleic acids are directly attached to said surface.

20. The method of claim 14, wherein said primers are attached directly to said surface.

21. The method of claim 14, wherein both said nucleic acids and said primers are attached directly to said surface.

22. The method of claim 14, wherein said nucleic acids are attached to said surface via a member of a binding pair.

23. The method of claim 22, wherein said member of a binding pair is selected from the group consisting of an antibody, and antigen, a receptor, and a ligand.

24. The method of claim 14, wherein said surface is an epoxide-coated surface.

25. The method of claim 24, wherein said epoxide coating is passivated to prevent non-specific binding.

26. The method of claim 25, wherein said surface is passivated with phosphate.

27. The method of claim 14, wherein said surface is glass or fused silica.

28. The method of claim 14, wherein said exposing and detecting steps are repeated at least once.

29. The method of claim 14, wherein the flow cell is operably positioned on a microscope stage such that the added nucleotides can be identified using the microscope.

30. The method of claim 29, wherein the nucleotides are identified using total internal reflection fluorescence.

31. The method of claim 14, wherein said optically labeled nucleotides are labeled with a fluorescent dye selected from the group consisting of fluorescein, rhodamine, cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof.

32. An apparatus comprising a flow cell having an inlet and an outlet, and a surface treated to allow attachment of nucleic acids thereto.

34. The apparatus of claim 32, wherein said epoxide is derivatized with a member of a binding pair.

35. The apparatus of claim 32, wherein said binding pair is selected from the group consisting of biotin/streptavidin and antibody/antigen.

36. The apparatus of claim 32 further comprising nucleic acids or primers attached to said surface such that at least a portion of said nucleic acids or primers are individually optically resolvable.

38. The apparatus of claim 35, wherein the nucleotides can be identified using total internal reflection fluorescence.

33. The apparatus of claim 33, wherein said surface comprises an epoxide or epoxide derivative.

37. The apparatus of claim 33, further comprising a microscope, wherein the flow cell can be operably positioned on a microscope stage such that the added nucleotides can be identified using the microscope.