METHODS AND APPARATUSES FOR ANALYZING POLYNUCLEOTIDE SEQUENCES

• US 20090253141A1
• Filed: 03/09/2009
• Published: 10/08/2009
• Est. Priority Date: 11/04/1999
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Quake, Stephen R.
• Application Number:
  US12/400,593
• Publication Number:
  US 20090253141A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00398 in multiple arrangements
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00628 Ionic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00632 Introduction of reactive groups to the surface
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 : pinch valves
• 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: B01L 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device

Abstract

Methods for high speed, high throughput analysis of polynucleotide sequences, and apparatuses with which to carry out the methods are provided in the invention.

Claims

1. A method of conducting single molecule sequencing, the method comprising:

providing a device comprising a synthesis channel or a reaction chamber,

introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;

conducting said reaction,

determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,

wherein one or more of the reagents is introduced in the synthesis channel or reaction chamber at a flow rate that does not cause substantial sheering of nucleic acid template and/or dislocation of the polymerase from nucleic acid complexes.

6. The method of claim 1, wherein the template is at least 1 kb in length.

7. The method of claim 1, wherein the template is at least about 20 kb in length.

12. The method of claim 1, further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate that does not cause substantial sheering of nucleic acid templates and/or dislocation of the polymerase from nucleic acid complexes.

17. The method of claim 1, wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera.

20. The method of claim 1, wherein the dye-labeled nucleotides are fluorescently labeled.

2. A method of conducting single molecule sequencing, the method comprising:

providing a device comprising a synthesis channel or a reaction chamber,

introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;

conducting said reaction,

determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,

wherein one or more of the reagents is introduced in the synthesis channel or reaction chamber at a flow rate that maintains substantially laminar flow in the channel or chamber.

8. The method of claim 2, wherein the template is at least 1 kb in length.

9. The method of claim 2, wherein the template is at least about 20 kb in length.

13. The method of claim 2, further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate that maintains substantially laminar flow in the channel or chamber.

18. The method of claim 2, wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera.

3. A method of conducting single molecule sequencing, the method comprising:

providing a device comprising a synthesis channel or a reaction chamber,

introducing in said synthesis channel or reaction chamber reagents necessary of a polymerase-mediated template-dependent nucleic synthesis reaction, comprising a plurality of templates, one or more primers, a polymerase and one or more dye-labeled nucleotides;

conducting said reaction,

determining a sequence of the template by detecting individual nucleotides incorporated into a copy of the template immobilized on a surface,

wherein one or more of the reagents is introduced into the synthesis channel or reaction chamber at a flow rate below 10 cm/sec.

4. The method of claim 3, wherein the flow rate is chosen from 0.1-1 cm/sec and 1-10 cm/sec.

5. The method of claim 4, wherein the flow rate is 0.1-1 cm/sec.

10. The method of claim 3, wherein the template is at least 1 kb in length.

11. The method of claim 3, wherein the template is at least about 20 kb in length.

14. The method of claim 3, further comprising washing unincorporated dye-labeled nucleotides away from the synthesis channel or reaction chamber at a flow rate below 10 cm/sec.

15. The method of claim 14, wherein the flow rate is chosen from 0.1-1 cm/sec and 1-10 cm/sec.

16. The method of claim 15, wherein the flow rate is 0.1-1 cm/sec.

19. The method of claim 3, wherein the method comprises a simultaneous detection of multiple regions on the surface using a CCD camera.