Bead emulsion nucleic acid amplification

• US 20080132693A1
• Filed: 10/31/2007
• Published: 06/05/2008
• Est. Priority Date: 01/29/2003
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 CORPORATION
• Inventors: Weiner, Michael P., Rothberg, Jonathan M., Lohman, Kenton L., Leamon, John H., Sarkis, Gary J., Srinivasan, Maithreyan, Berka, Jan, Lefkowitz, Steve, Chen, Yi-Ju, Makhijani, Vinod B.
• Granted Patent: US 8,012,690 B2
• Time in Patent Office: Days
• US Class Current: 536/25.4
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0877 Flow chambers
• 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 3/502715 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: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 : Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2531/125 : Rolling circle
• CPC Class Codes: C12Q 2535/119 : Double strand sequencing
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/149 : Sequential reactions
• CPC Class Codes: C12Q 2547/101 : by confinement to a single tube/container
• CPC Class Codes: C12Q 2547/107 : Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/119 : based on extraction of label to an organic phase, i.e. partitioning of label between different organic phases
• CPC Class Codes: C12Q 2565/301 : Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: G01N 2021/6484 : Optical fibres
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• 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 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01R 33/1269 : of molecules labeled with magnetic beads magnetic particles for bio assay G01N33/54326
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

Claims

1-44. -44. (canceled)

45. A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:

distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;

amplifying the species of template nucleic acid molecules within the first subset of droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of aqueous droplets;

breaking the aqueous emulsion droplets to release the beads of the first and second subsets; and

attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads enabled for isolation under a selective condition; and

applying the selective condition to isolate the one or more enrichment beads from the beads of the second subset.

46. The method of claim 45, wherein:

the solution comprises a ratio of a number of the template nucleic acid molecules to a number of the beads selected from a group consisting of a ratio that is equal to about 1, a ratio that is less than about 1, and a ratio that is about 0.1.

47. The method of claim 45, wherein:

the solution further comprises a sufficient mixture of reagents necessary for performing an amplification reaction of the species of template nucleic acid molecule.

48. The method of claim 45, wherein:

the step of amplifying employs a polymerase chain reaction amplification process.

49. The method of claim 48, wherein:

the complementary nucleic acid molecules are immobilized to the beads of the first subset during the polymerase chain reaction amplification process by hybridizing a plurality of copies of the species of template nucleic acid molecules to a plurality of primers immobilized on the beads and extending the complementary nucleic acid molecules from the immobilized primers.

50. The method of claim 45, wherein:

the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer disposed on the enrichment beads to a portion of one or more of the immobilized complementary nucleic acid molecules.

51. The method of claim 50, wherein:

the capture primer is complementary to a 3′

portion common to the immobilized complementary nucleic acid molecules wherein the 3′

portion comprises a sequence length selected from the group consisting of at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, and at least 40 bases.

52. The method of claim 45, wherein:

the selective condition comprises a magnetic field enabled to isolate the one or more enrichment beads from the beads of the second subset.

53. The method of claim 45, further comprising:

removing the isolated enrichment beads comprising the attached beads of the first subset; and

releasing the beads of the first subset comprising the immobilized complementary nucleic acid molecules from the one more enrichment beads.

54. The method of claim 53, further comprising:

determining a nucleic acid composition for the one or more species of nucleic acid molecules from the immobilized complementary nucleic acid molecules.

55. The method of claim 45, further comprising:

reusing the beads of the second subset in a subsequent amplification and enrichment process.

56. A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:

distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise at least one of the beads and one or more species of the template nucleic acid molecules compartmentalized therein, and a second subset of the droplets comprise at least one of the beads without any of the species of template nucleic acid molecules compartmentalized therein;

amplifying the species of template nucleic acid molecules within the first subset of droplets, wherein during the amplification a plurality of copies of the species of template nucleic acid molecule hybridize to a plurality of primers immobilized on the one or more beads and a plurality of complementary nucleic acid molecules are extended therefrom;

breaking the aqueous emulsion droplets to release the beads of the first and second subsets;

disassociating the hybridized copies of the species of the template nucleic acid molecules from the primers and extended complementary nucleic acid molecules immobilized on the beads of the first subset;

hybridizing the 3′

end of one or more of the complementary nucleic acid molecules immobilized on the beads of the first subset to a second primer disposed on one or more enrichment beads enabled for isolation under a selective condition thereby linking the enrichment beads to one or more of the beads of the first subset; and

applying the selective condition to isolate the one or more enrichment beads from the beads of the second subset.

57. The method of claim 56, wherein:

the solution comprises a ratio of a number of the template nucleic acid molecules to a number of the beads selected from a group consisting of a ratio that is equal to about 1, a ratio that is less than about 1, and a ratio that is about 0.1.

58. The method of claim 56, wherein:

the second primer comprises a binding moiety that binds to a capture moiety immobilized on the enrichment beads.

59. The method of claim 58, wherein:

the binding moiety comprises a biotin moiety and the capture moiety comprises a streptavidin moiety.

60. The method of claim 56, wherein:

the solution further comprises a sufficient mixture of reagents necessary for performing an amplification reaction of the species of template nucleic acid molecule.

61. The method of claim 56, further comprising:

extending the second primer hybridized to the 3′

end of the one or more complementary nucleic acid molecules, wherein the extension enhances bonding between the second primer and the complementary nucleic acid molecules immobilized on the beads of the first subset.

62. The method of claim 56, wherein:

the 3′

end of the immobilized complementary nucleic acid molecules enabled to hybridize the second primer comprises a sequence length selected from the group consisting of at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, and at least 40 bases.

63. The method of claim 56, wherein:

the selective condition comprises a magnetic field enabled to isolate the one or more enrichment beads from the beads of the second subset.

64. The method of claim 56, further comprising:

removing the isolated enrichment beads comprising the attached beads of the first subset; and

releasing the beads of the first subset comprising the immobilized complementary nucleic acid molecules from the one more enrichment beads.

65. The method of claim 64, further comprising:

determining a nucleic acid composition for the one or more species of nucleic acid molecules from the immobilized complementary nucleic acid molecules.

66. The method of claim 56, further comprising:

reusing the beads of the second subset in a subsequent amplification and enrichment process.

67. A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:

distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise at least one of the beads and one or more species of the template nucleic acid molecules compartmentalized therein, and a second subset of the droplets comprise at least one of the beads without any of the species of template nucleic acid molecules compartmentalized therein;

amplifying the species of template nucleic acid molecules within the first subset of droplets, wherein during the amplification a plurality of copies of the species of template nucleic acid molecule hybridize to a plurality of primers immobilized on the one or more beads and a plurality of complementary nucleic acid molecules are extended therefrom;

breaking the aqueous emulsion droplets to release the beads of the first and second subsets;

disassociating the hybridized copies of the species of the template nucleic acid molecules from the primers and extended complementary nucleic acid molecules immobilized on the beads of the first subset;

hybridizing the 3′

end of one or more of the complementary nucleic acid molecules immobilized on the beads of the first subset to a second primer comprising a binding moiety;

attaching the binding moiety of the hybridized second primer to a capture moiety disposed upon a substrate thereby immobilizing the first subset of beads on the substrate.

68. The method of claim 67, further comprising:

flowing the beads of the first and second subsets in a solution over the substrate, wherein the beads of the second subset remain in the solution.

69. The method of claim 67, wherein:

the substrate comprises an enrichment bead enabled for isolation under a selective condition.