Bead emulsion nucleic acid amplification

• US 8,748,102 B2
• Filed: 02/23/2011
• Issued: 06/10/2014
• 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: Berka, Jan, Chen, Yi-Ju, Leamon, John H., Lefkowitz, Steve, Lohman, Kenton L., Makhijani, Vinod B., Rothberg, Jonathan M., Sarkis, Gary J., Srinivasan, Maithreyan, Weiner, Michael P.
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US13/033,240
• Publication Number:
  US 20110201526A1
• Time in Patent Office: 1,203 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 analyzing nucleic acid sequences comprising:

(a) generating a plurality of molecules of a fragment of deoxyribonucleic acid;

(b) delivering the plurality of molecules of the fragment of deoxyribonucleic acid into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single molecule of the fragment of deoxyribonucleic acid, a single bead capable of hybridizing the fragment of deoxyribonucleic acid, and reagents necessary to perform deoxyribonucleic acid amplification;

(c) amplifying the fragment of deoxyribonucleic acid in the microreactors to form amplified copies of said fragment of deoxyribonucleic acid bound to beads in the microreactors;

(d) determining the presence of amplified copies of said fragment of deoxyribonucleic acid bound to a bead.

2. The method of claim 1 wherein step (c) is accomplished using polymerase chain reaction.

14. The method of claim 1, wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads.

3. A method for analyzing nucleotide sequences from a biological sample obtained from an animal, plant or fungus, comprising:

forming microemulsions comprising one or more species of analyte DNA molecules, such that a plurality of aqueous compartments comprise a single species of analyte DNA;

amplifying analyte DNA molecules in the microemulsions in the presence of reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of the single species of analyte DNA molecule;

separating the product beads from analyte DNA molecules which are not bound to product beads;

determining the presence of the single species of analyte DNA molecule which is bound to the product beads.

5. The method of claim 3, wherein prior to the step of separating, the microemulsions are broken by addition of one or more detergents.

6. The method of claim 3, wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.

7. The method of claim 3, wherein the analyte DNA molecules are genomic DNA.

8. The method of claim 3, wherein the analyte DNA molecules are PCR products made from genomic DNA.

9. The method of claim 3, wherein the analyte DNA molecules are derived from a single individual.

10. The method of claim 3, wherein the reagent beads are magnetic.

11. The method of claim 3, wherein the single species of analyte DNA molecules are DNA fragments.

15. The method of claim 3, wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads.

4. A method for analyzing nucleotide sequences comprising:

forming microemulsions comprising one or more species of analyte DNA molecules;

amplifying analyte DNA molecules in the microemulsions in the presence of reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of one species of analyte DNA molecule, wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads;

separating the product beads from analyte DNA molecules which are not bound to product beads;

determining the presence of the one species of analyte DNA molecule which is bound to the product beads.

12. The method of claim 4, wherein the one or more species of analyte DNA molecules are a single species of analyte DNA molecules.

13. The method of claim 4, wherein the one or more species of analyte DNA molecules are DNA fragments.