Method and Compositions for Detection and Enumeration of Genetic Variations
First Claim
1. A method for sequencing nucleic acids comprising:
- (a) fragmenting genomic nucleic acid molecules to generate a plurality of fragmented nucleic acids;
(b) delivering the fragmented nucleic acids into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a fragmented nucleic acid, a single bead capable of hybridizing to the fragmented nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification;
(c) amplifying the fragmented nucleic acids in the microreactors to form amplified copies of said nucleic acids and hybridizing the amplified copies to beads in the microreactors;
(d) delivering the beads to an array of reaction chambers, wherein a plurality of the reaction chambers comprise no more than a single nucleic acid bound bead; and
(e) performing a sequencing reaction simultaneously on a plurality of the reaction chambers.
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Abstract
Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently-labeled particles via flow cytometry. Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and employed for further experimentation. This approach can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.
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4 Claims
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1. A method for sequencing nucleic acids comprising:
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(a) fragmenting genomic nucleic acid molecules to generate a plurality of fragmented nucleic acids; (b) delivering the fragmented nucleic acids into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single copy of a fragmented nucleic acid, a single bead capable of hybridizing to the fragmented nucleic acid, and amplification reaction solution containing reagents necessary to perform nucleic acid amplification; (c) amplifying the fragmented nucleic acids in the microreactors to form amplified copies of said nucleic acids and hybridizing the amplified copies to beads in the microreactors; (d) delivering the beads to an array of reaction chambers, wherein a plurality of the reaction chambers comprise no more than a single nucleic acid bound bead; and (e) performing a sequencing reaction simultaneously on a plurality of the reaction chambers. - View Dependent Claims (2, 3, 4)
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Specification