Bead emulsion nucleic acid amplification
First Claim
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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.
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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.
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Citations
44 Claims
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1. A method for amplifying one or more nucleic acids comprising the steps of:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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- 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.
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22. A method for amplifying a nucleic acid comprising the steps of:
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(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.
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34. A method for producing a clonal population of nucleic acids, comprising:
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(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. - View Dependent Claims (35, 36, 37, 38)
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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.
- View Dependent Claims (40, 41, 42, 43, 44)
Specification