Bead emulsion nucleic acid amplification

• US 20050079510A1
• Filed: 01/28/2004
• Published: 04/14/2005
• 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 7,842,457 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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. A method for amplifying one or more nucleic acids comprising the steps of:

(a) forming a water-in-oil emulsion to create a plurality of aqueous microreactors wherein at least one of the microreactors comprises a single nucleic acid template, a single bead capable of binding to the nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification;

(b) amplifying the nucleic acids in the microreactors to form amplified copies of said nucleic acids; and

(c) binding the amplified copies to the beads in the microreactors.

2. The method of claim 1, wherein a majority of the microreactors include a single nucleic acid.

3. The method of claim 1, wherein said amplification reaction solution is a polymerase chain reaction solution comprising nucleotide triphosphates, a thermostable polymerase, and nucleic acid primers suspended in a buffer compatible with polymerase chain reaction conditions.

4. The method of claim 3, wherein said polymerase chain reaction is an asymmetric polymerase chain reaction.

5. The method of claim 3, wherein said polymerase chain reaction is a symmetric polymerase chain reaction.

6. The method of claim 1, wherein said emulsion additionally contains emulsion stabilizers.

7. The method of claim 6, wherein said emulsion stabilizers are selected from the group consisting of Atlox 4912, Span 80, and combinations and mixtures thereof.

8. The method of claim 1 wherein said emulsion is heat stable.

9. The method of claim 8 wherein said emulsion is heat stable to 95°

C.

10. The method of claim 1, wherein amplification is carried out by a method selected from the group consisting of transcription-based amplification, rapid amplification of cDNA ends, continuous flow amplification, and rolling circle amplification.

11. The method of claim 1, wherein the emulsion is formed by the dropwise addition of the nucleic acid templates, beads, and amplification reaction solution into an oil.

12. The method of claim 1, performed with at least 10,000 nucleic acids.

13. The method of claim 1, performed with at least 50,000 nucleic acids.

14. The method of claim 1, wherein the microreactors have an average size of 50 to 250 μ

m in diameter.

15. The method of claim 1, wherein after step (c) each bead captures more than 10,000 amplification copies of a nucleic acid template.

23. A kit for conducting nucleic acid amplification of a nucleic acid template comprising:

(a) a nucleic acid capture bead;

(b) an emulsion oil;

(c) one or more emulsion stabilizers;

(d) instructions for performing the method of claim 1 or claim 22.

24. The method of claim 1 or claim 22 further comprising the step of enriching for beads which bind amplified copies of the nucleic acid away from beads to which no nucleic acid is bound, the enrichment step selected from the group consisting of affinity purification, electrophoresis and cell sorting.

25. The method of claim 24 wherein the enrichment step is performed by affinity purification with magnetic beads that bind nucleic acid.

26. The method of claim 1 or 22, wherein at least 100,000 copies of each target nucleic acid molecule are bound to each bead.

27. The method of claim 1 or 22, wherein at least 1,000,000 copies of each target nucleic acid molecule are bound to each bead.

28. The method of claim 1 or 22, wherein between at least 1 to 20,000,000 copies of each target nucleic acid molecule are bound to each bead.

29. The method of claim 1 or 22, wherein the beads are sepharose beads.

30. The method of claim 1 or 22, wherein amplified copies are bound to the beads by a binding pair selected from the group consisting of antigen/antibody, ligand/receptor and polyhistidine/nickel.

31. The method of claim 30, wherein the binding pair is avidin/biotin.

32. The method of claim 25, further comprising the steps of:

separating the template carrying beads and magnetic bead; and

removing the magnetic beads with a magnetic field.

33. The method of claim 32, wherein the separating is achieved by incubation at a temperature greater than 45°

C. or by incubating the template carrying beads and the magnetic beads in a solution with a basic pH.

16. A library comprising a plurality of nucleic acid molecules, wherein each nucleic acid molecule is separately immobilized to a different bead, and wherein each bead comprises over 1,000,000 clonal amplification copies of each nucleic acid molecule, wherein the library is contained in a single vessel.

17. The library of claim 16, wherein the nucleic acid molecules are selected from the group consisting of genomic DNA, cDNA, episomal DNA, BAC DNA, and YAC DNA.

18. The library of claim 16, wherein the genomic DNA is selected from the group consisting of animal, plant, viral, bacterial, and fungal genomic DNA.

19. The library of claim 18, wherein the genomic DNA is human genomic DNA or human cDNA.

20. The library of claim 16, wherein the bead has a diameter of 2 microns to 100 microns.

21. The library of claim 16, wherein the bead is a sepharose bead.

22. A method for amplifying a nucleic acid comprising the steps of:

(a) providing a nucleic acid template to be amplified;

(b) providing a solid support material comprising a generally spherical bead having a diameter about 10 to about 80 μ

m, wherein the bead is capable of binding to the nucleic acid template;

(c) mixing the nucleic acid template and the bead in an amplification reaction solution containing reagents necessary to perform a nucleic acid amplification reaction in a water-in-oil emulsion;

(d) amplifying the nucleic acid template to form amplified copies of said nucleic acid template; and

(e) binding the amplified copies to the bead.

34. A method for producing a clonal population of nucleic acids, comprising:

(a) providing a plurality of nucleic acid templates from 50-800 bp in length and beads capable of binding to the nucleic acid templates;

(b) mixing the nucleic acid templates and the beads in a biological reaction solution containing reagents necessary to amplify the nucleic acid templates;

(c) forming an emulsion to create a plurality of microreactors comprising the nucleic acid templates, beads, and biological reaction solution, wherein at least one of the microreactors comprises a single nucleic acid template and a single bead encapsulated in the biological reaction solution, wherein the microreactors are contained in the same vessel.

35. The method of claim 34 further comprising transcribing and translated the nucleic acids to generate at least 10,000 copies of an expression product.

36. The method of claim 35, wherein said expression product is bound to said beads by a binding pair selected from the group consisting of antigen/antibody, ligand/receptor, 6×

his/nickel-nitrilotriacetic acid, and FLAG tag/FLAG antibody binding pairs.

37. The method of claim 35, wherein the method produces a clonal population of proteins.

38. The method of claim 37, wherein the proteins are selected from the group consisting of antibodies, antibodies fragments, and engineered antibodies.

39. An emulsion comprising a plurality of thermostable microreactors, wherein the microreactors are 50 to 200 μ

m in diameter and comprise a biological reaction solution.

40. The emulsion of claim 39, wherein the biological reaction solution comprises reagents for performing polymerase chain reaction amplification.

41. The emulsion of claim 39, wherein the biological reaction solution comprises reagents for performing coupled transcription and translation reactions.

42. The emulsion of claim 40 or claim 41, wherein the plurality of microreactors comprise a nucleic acid template.

43. The emulsion of claim 42, wherein the plurality of microreactors comprise one or fewer nucleic acid templates.

44. The emulsion of claim 43, wherein the plurality of microreactors comprise one or fewer beads that bind to the nucleic acid templates.