Methods and apparatuses for analyzing polynucleotide sequences

• US 20060019263A1
• Filed: 12/01/2004
• Published: 01/26/2006
• Est. Priority Date: 06/28/1999
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen, Kartalov, Emil Paskalev
• Granted Patent: US 7,501,245 B2
• 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 for performing a nucleic acid sequencing reaction, the method comprising the steps of:

providing a substrate comprising

a poly(dimethylsiloxane) layer grafted to poly(ethylene glycol); and

a polyelectrolyte multilayer linked to said poly(dimethylsiloxane) by said poly(ethylene glycol); and

a polynucleotide attached to said substrate; and

exposing said polynucleotide to a mixture comprising a polymerase and a primer and a nucleotide under conditions wherein said nucleotide is capable of incorporation into said primer.

2. The method of claim 1 wherein a plurality of polynucleotides are attached to said substrate such that each polynucleotide is individually optically resolvable.

3. The method of claim 2 wherein said nucleotide comprises an optically detectable label.

4. The method of claim 3, further comprising the step of determining the incorporation of said nucleotide into said primer.

5. The method of claim 4, further comprising the step of repeating said exposure and determining step with a second nucleotide.

6. The method of claim 1 wherein said poly(ethylene glycol) is diacrylated poly(ethylene glycol).

7. The method of claim 2 wherein said label comprises a fluorophore.

8. A surface for nucleic acid sequencing, said surface comprising a plurality of chemical layers including a poly(dimethylsiloxane) layer, a poly(ethylene glycol) layer, a polyelectrolyte multilayer, a nucleic acid linkage layer and a nucleic acid layer,

wherein said poly(ethylene glycol) layer is positioned between said poly(dimethylsiloxane) layer and said polyelectrolyte multilayer, and

wherein said nucleic acid linkage layer attaches said nucleic acid layer to said polyelectrolyte multilayer.

9. The surface of claim 8 wherein said nucleic acid layer comprises a plurality of polynucleotides and wherein each polynucleotide is individually optically resolvable.

10. The surface of claim 9 wherein said nucleic acid linkage layer comprises biotin-streptavidin or digoxygenin-antidigoxygenin.

11. The surface of claim 8 wherein said poly(ethylene glycol) is diacrylated poly(ethylene glycol).

12. A method for performing a nucleic acid sequencing reaction, the method comprising

exposing a substrate-bound polynucleotide to a mixture comprising a polymerase and a primer and a nucleotide under conditions wherein said nucleotide is capable of incorporation into said primer,

wherein said substrate comprises

a poly(dimethylsiloxane) layer grafted to poly(ethylene glycol);

and

polyelectrolyte multilayer linked to said poly(dimethylsiloxane) by said poly(ethylene glycol).

13. The method of claim 12 wherein a plurality of polynucleotides are attached to said substrate such that each polynucleotide is individually optically resolvable.

14. The method of claim 13 wherein said nucleotide comprises an optically detectable label.

15. The method of claim 14, further comprising the step of determining the incorporation of said nucleotide into said primer.

16. The method of claim 15, further comprising the step of repeating said exposure and determining step with a second nucleotide.

17. The method of claim 12 wherein said poly(ethylene glycol) is diacrylated poly(ethylene glycol).

18. The method of claim 13 wherein said label comprises a fluorophore.

19. The method of claim 16, further comprising the step of serially repeating said exposure and determining step with a third nucleotide and a fourth nucleotide.

20. The method of claim 12 wherein said mixture comprises four species of nucleotides simultaneously.