CA2513535A1 - Bead emulsion nucleic acid amplification - Google Patents
Bead emulsion nucleic acid amplification Download PDFInfo
- Publication number
- CA2513535A1 CA2513535A1 CA002513535A CA2513535A CA2513535A1 CA 2513535 A1 CA2513535 A1 CA 2513535A1 CA 002513535 A CA002513535 A CA 002513535A CA 2513535 A CA2513535 A CA 2513535A CA 2513535 A1 CA2513535 A1 CA 2513535A1
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- Prior art keywords
- nucleic acid
- bead
- beads
- emulsion
- microreactors
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract 48
- 108020004707 nucleic acids Proteins 0.000 title claims abstract 48
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract 48
- 239000011324 bead Substances 0.000 title claims abstract 37
- 230000003321 amplification Effects 0.000 title claims abstract 17
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract 17
- 239000000839 emulsion Substances 0.000 title claims 16
- 238000000034 method Methods 0.000 claims abstract 35
- 238000006243 chemical reaction Methods 0.000 claims abstract 13
- 238000003752 polymerase chain reaction Methods 0.000 claims 6
- 239000003153 chemical reaction reagent Substances 0.000 claims 5
- 239000002299 complementary DNA Substances 0.000 claims 3
- 239000003381 stabilizer Substances 0.000 claims 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims 2
- 229920002684 Sepharose Polymers 0.000 claims 2
- 238000001261 affinity purification Methods 0.000 claims 2
- 239000000427 antigen Substances 0.000 claims 2
- 102000036639 antigens Human genes 0.000 claims 2
- 108091007433 antigens Proteins 0.000 claims 2
- 239000003446 ligand Substances 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 claims 2
- 102000004169 proteins and genes Human genes 0.000 claims 2
- 238000013518 transcription Methods 0.000 claims 2
- 230000035897 transcription Effects 0.000 claims 2
- 239000007762 w/o emulsion Substances 0.000 claims 2
- 108090001008 Avidin Proteins 0.000 claims 1
- 241001465754 Metazoa Species 0.000 claims 1
- 238000012408 PCR amplification Methods 0.000 claims 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims 1
- 238000002299 affinity electrophoresis Methods 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 claims 1
- 229960002685 biotin Drugs 0.000 claims 1
- 235000020958 biotin Nutrition 0.000 claims 1
- 239000011616 biotin Substances 0.000 claims 1
- 239000012634 fragment Substances 0.000 claims 1
- 230000002538 fungal effect Effects 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 229910052759 nickel Inorganic materials 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- -1 nucleotide triphosphates Chemical class 0.000 claims 1
- 229920002704 polyhistidine Polymers 0.000 claims 1
- 238000005096 rolling process Methods 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 239000001226 triphosphate Substances 0.000 claims 1
- 235000011178 triphosphate Nutrition 0.000 claims 1
- 230000003612 virological effect Effects 0.000 claims 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- B01L7/00—Heating or cooling apparatus; Heat insulating devices
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
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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 (45)
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.
(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.
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 pm 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.
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 µm, 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.
(b) providing a solid support material comprising a generally spherical bead having a diameter about 10 to about 80 µm, 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.
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.
(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.
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.
(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, 6Xhis/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 µm 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 44. The emulsion of claim 43, wherein the plurality of microreactors comprise one or fewer beads that bind to the nucleic acid templates.
45
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- 2016-06-23 US US15/190,886 patent/US10240192B2/en not_active Expired - Lifetime
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2019
- 2019-01-16 US US16/249,681 patent/US10982274B2/en not_active Expired - Lifetime
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8048627B2 (en) | 2003-07-05 | 2011-11-01 | The Johns Hopkins University | Method and compositions for detection and enumeration of genetic variations |
US9328343B2 (en) | 2003-07-05 | 2016-05-03 | The Johns Hopkins University | Method and compositions for detection and enumeration of genetic variations |
US10604797B2 (en) | 2003-07-05 | 2020-03-31 | The Johns Hopkins University | Method and compositions for detection and enumeration of genetic variations |
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