METHODS AND APPLICATIONS FOR STITCHED DNA BARCODES
First Claim
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1. A method of determining the presence of at least one genetic mutation in a population of organisms, comprising the steps of:
- (a) creating one or more aqueous emulsion droplets, wherein at least one emulsion droplet includes an organism having at least one barcode-associated mutation;
(b) performing a polymerase chain reaction (PCR) in the emulsion droplet to amplify at least one fused DNA product of one or more barcodes;
(c) recovering the aqueous phase from the emulsion droplet, wherein the aqueous phase includes the amplified, barcode-associated mutation; and
(d) detecting the presence of the amplified, barcode-associated mutation.
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Abstract
Methods of identifying the genotypes of a plurality of single cells, wherein each cell includes a plurality of DNA barcodes, each associated with a genetic mutation or marker, are provided. In particular, methods including linking a plurality of DNA barcodes together to create a stitched barcode, amplifying the stitched barcode and sequencing the stitched barcode are provided. Also provided are methods of determining the presence of at least one genetic mutation in a population of cells.
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Citations
27 Claims
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1. A method of determining the presence of at least one genetic mutation in a population of organisms, comprising the steps of:
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(a) creating one or more aqueous emulsion droplets, wherein at least one emulsion droplet includes an organism having at least one barcode-associated mutation; (b) performing a polymerase chain reaction (PCR) in the emulsion droplet to amplify at least one fused DNA product of one or more barcodes; (c) recovering the aqueous phase from the emulsion droplet, wherein the aqueous phase includes the amplified, barcode-associated mutation; and (d) detecting the presence of the amplified, barcode-associated mutation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of identifying the genotype of an organism, wherein the organism comprises a plurality of barcode-associated mutations, comprising the steps of:
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(a) covalently linking a plurality of barcode-associated mutations together to create a stitched barcode; (b) amplifying the stitched barcode; and (c) determining the identity of the stitched barcode. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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22. A method of making a library of organisms each organism carrying a combination of barcode-associated mutations, comprising the steps of:
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(a) providing a first population of organisms each of which contains at least one of a first plurality of barcode-associated mutations; (b) providing a second population of organisms each of which contains at least one of a second plurality of barcode-associated mutations; and (c) combining the first population of organisms and the second population of organisms such that at least one resulting organism comprises a combination of at least one of the first plurality of barcode-associated mutations and at least one of the second plurality of barcode-associated mutations. - View Dependent Claims (23, 24, 25, 26, 27)
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Specification