System and method for improved signal detection in nucleic acid sequencing

• US 20090203086A1
• Filed: 01/29/2009
• Published: 08/13/2009
• Est. Priority Date: 02/06/2008
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Chen, Zhoutao, Kebbel, Andrew Daniel, Chen, Yi-Ju, Kim, Jong-Bum, Nealis, James Matthew
• Application Number:
  US12/322,284
• Publication Number:
  US 20090203086A1
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00391 Rotary valves
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: G01N 2021/0325 Cells for testing reactions, e.g. containing reagents
• CPC Class Codes: G01N 21/03 Cuvette constructions

Abstract

An embodiment of a system for reducing crosstalk in a parallel sequencing platform is described that comprises a substrate with a plurality of individual reaction environments that include a species of nucleic acid template, and a plurality of spatially localized reactants, wherein the localized reactants minimize the transmission of reaction products to a neighboring reaction environment due to a relative position of the localized reactants in the reaction environment.

Claims

1. A system for reducing crosstalk in a parallel sequencing platform, comprising:

a substrate comprising a plurality of individual reaction environments that include a species of nucleic acid template, and a plurality of spatially localized reactants, wherein the localized reactants minimize the transmission of reaction products to a neighboring reaction environment due to a relative position of the localized reactants in the reaction environment.

2. The system of claim 1, wherein:

the substrate comprises a fiber optic faceplate, where the reaction environments comprise wells etched into a first surface of the fiber optic faceplate.

3. The system of claim 1, wherein:

the spatially localized reactants are disposed on a solid phase substrate.

4. The system of claim 3, wherein:

the solid phase substrate comprises a bead substrate.

5. The system of claim 1, wherein:

the species of nucleic acid template comprises a population of substantially identical copies of a nucleic acid template molecule, wherein the substantially identical copies are disposed on a solid phase substrate.

6. The system of claim 5, wherein:

the solid phase substrate comprises a bead substrate.

7. The system of claim 1, wherein:

the spatially localized regents comprise a first class of reagent and a second class of reagent.

8. The system of claim 7, wherein:

the first class of reagent is selected from the group consisting of pyrophosphatase and apyrase.

9. The system of claim 8 wherein:

the pyrophosphatase removes 90%-99% of pyrophosphate that diffuses into and out of the reaction environment.

10. The system of claim 7, wherein:

the second class of reagent is selected from the group consisting of sulfurylase and luciferase.

11. The system of claim 7, wherein:

the individual reaction environments comprise wells, wherein the first class reagent is localized to a top region of the well and the second class of reagent is localized to a bottom region of the well.

12. The system of claim 11, wherein:

the species of nucleic acid template is disposed on a solid phase substrate localized to a central region of the well, wherein the central region further comprises a plurality of solid phase substrates that are free of reactants and occupy space within the central region.

13. The system of claim 12, wherein:

the nucleic acid template substrate comprises a bead and the reactant-free substrates comprise beads, wherein the nucleic acid template bead and the non-reactant beads are compacted in the central region of the well.

14. The system of claim 13, wherein:

the nucleic acid template bead and the non-reactant beads are compacted by centrifugal force.

15. A method for reducing crosstalk in a parallel sequencing platform, comprising:

providing a substrate comprising a plurality of individual reaction environments that include a species of nucleic acid template, and a plurality of spatially localized reactants; and

exposing the reaction environments to a nucleotide species to produce a detectable signal within one or more of the reaction environments, wherein the localized reactants within the reaction environments minimize the transmission of reaction products to a neighboring reaction environment.

16. The system of claim 15, wherein:

the substrate comprises a fiber optic faceplate, where the reaction environments comprise wells etched into a first surface of the fiber optic faceplate.

17. The method of claim 15, wherein:

the spatially localized reactants are disposed on a solid phase substrate.

18. The method stem of claim 17, wherein:

the solid phase substrate comprises a bead substrate.

19. The system of claim 15, wherein:

the species of nucleic acid template comprises a population of substantially identical copies of a nucleic acid template molecule, wherein the substantially identical copies are disposed on a solid phase substrate.

20. The system of claim 19, wherein:

the solid phase substrate comprises a bead substrate.

21. The method of claim 15, wherein:

the spatially localized regents comprise a first class of reagent and a second class of reagent.

22. The method of claim 21, wherein:

the first class of reagent is selected from the group consisting of pyrophosphatase and apyrase.

23. The method of claim 22 wherein:

the pyrophosphatase removes 90%-99% that diffuses into and out of the reaction environment.

24. The method of claim 21, wherein:

the second class of reagent is selected from the group consisting of sulfurylase and luciferase.

25. The method of claim 21, wherein:

the individual reaction environments comprise wells, wherein the first class reagent is localized to a top region of the well and the second class of reagent is localized to a bottom region of the well.

26. The method of claim 25, wherein:

the nucleic acid template is disposed on a solid phase substrate localized to a central region of the well, wherein the central region further comprises a plurality of solid phase substrates that are free of reactants and occupy space within the central region.

27. The method of claim 26, wherein:

the nucleic acid template substrate comprises a bead and the reactant-free substrates comprises beads, wherein the nucleic acid template bead and the non-reactant beads are compacted in the central region of the well.

28. The method of claim 27, wherein:

the nucleic acid template bead and the non-reactant beads are compacted by centrifugal force.

29. A kit for performing the method of claim 15, comprising:

the substrate comprising a fiber optic faceplate;

the localized reactants comprising a first class of reagent sequestered to a plurality of beads and a second class of reagent sequestered to a plurality of beads; and

a plurality of non-reactant beads.

30. The kit of claim 29, wherein:

the first class of reagent is selected from the group consisting of pyrophosphatase and apyrase.

31. The kit of claim 29, wherein:

the second class of reagent is selected from the group consisting of sulfurylase and luciferase.