Bead emulsion nucleic acid amplification

• US 7,842,457 B2
• Filed: 01/28/2004
• Issued: 11/30/2010
• Est. Priority Date: 01/29/2003
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• 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.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US10/767,899
• Publication Number:
  US 20050079510A1
• Time in Patent Office: 2,498 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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 onto a bead comprising the steps of:

(a) forming a water-in-oil emulsion to create a plurality of aqueous microreactors wherein a plurality of the microreactors comprises on average one single stranded nucleic acid template, a single bead with a first population comprising a plurality of molecules of a first primer species disposed thereon, and an amplification reaction solution comprising a second population comprising a plurality of molecules of the first primer species and a plurality of molecules of a second primer species and reagents necessary to perform nucleic acid amplification, wherein the first primer species is capable of binding to the single stranded nucleic acid template, the second primer species is capable of binding to a complementary strand of the single stranded nucleic acid template, and further wherein the molecules of the second primer species and the molecules of the first population of the primer species are each present in greater numbers within the aqueous microreactors than the number of molecules of the second population of the first primer species;

(b) asymmetrically amplifying the single stranded nucleic acid template and the complementary strand to the template strand in the amplification reaction solution to form a population of amplified copies of the single stranded template nucleic acid, wherein substantially all of the molecules of the second population of the first primer species in the amplification reaction solution are depleted;

(c) binding a plurality of the asymmetrically amplified copies of the single stranded template nucleic acid to the first population of the first primer species on the bead in a plurality of the microreactors, wherein at least 100,000 bead bound complementary strands are extended from the first primer species to form a population of beads with amplified nucleic acid template bound thereto;

(d) breaking the emulsion to release the population of beads with amplified nucleic acid template bound thereto from the microreactors and away from the amplification reaction solution comprising unbound amplification products; and

(e) enriching for beads with amplified nucleic acid template bound thereto by removing beads to which no nucleic acid is bound; and

(f) distributing the beads with amplified nucleic acid template bound thereto onto an array.

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 further comprising nucleotide triphosphates, a thermostable polymerase, and a buffer compatible with polymerase chain reaction conditions.

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

5. The method of claim 4, wherein said emulsion stabilizers are selected from the group consisting of Atlox 4912, Span 80, and combinations and mixtures thereof.

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

7. The method of claim 6 wherein said emulsion is heat stable to 95°

C.

8. 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.

9. 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.

10. The method of claim 1, performed with at least 10,000 nucleic acid templates.

11. The method of claim 1, performed with at least 50,000 nucleic acid templates.

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

m.

13. The method of claim 1, wherein each bead binds more than 10,000 asymmetrically amplified copies of the single stranded nucleic acid template.

14. The method of claim 1, further comprising the step of:

(g) sequencing the amplified nucleic acid templates.

15. The method of claim 1 wherein enrichment step (f) is performed using magnetic beads having primers attached thereto that bind the amplified nucleic acid template.

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

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

18. The method of claim 1, wherein the beads are sepharose beads.

19. The method of claim 15, further comprising, after step (e), the step of:

separating the beads with amplified nucleic acid template thereon away from the magnetic beads.

20. The method of claim 19, wherein the separating is achieved by incubation at a temperature greater than 45°

C. or by incubating the beads with amplified nucleic acid template thereon and the magnetic beads in a solution with a basic pH.