DIGITAL PCR BARCODING
1 Assignment
0 Petitions
Accused Products
Abstract
Methods, compositions, and kits are provided for nucleic acid analysis, including single cell analysis.
215 Citations
135 Claims
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1-104. -104. (canceled)
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105. A method of generating a plurality of partition-specific barcoded partitions, the method comprising:
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(a). conjugating a reverse amidite nucleotide to a plurality of precursor particles, wherein the conjugating is performed in at least four separate reactions, each reaction conjugating a different reverse amidite nucleotide, and wherein after the conjugating, the particles are combined and mixed; (b). repeating (a) from 6-20 times, thereby generating a plurality of barcoded particles, wherein the individual barcoded particles comprise oligonucleotides comprising a plurality of copies of a particle-specific barcode, wherein the individual oligonucleotides comprise a 5′ and
a 3′
end, the oligonucleotides are conjugated to the particles at the 5′
end, and the 3′
end is available for ligation and/or extension by a polymerase; and(c). partitioning the barcoded particles, thereby generating a plurality of partition-specific barcoded partitions. - View Dependent Claims (106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 126, 133)
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109. The method of claim 109, wherein the capture region comprises a randomer, a poly-thymine, or a poly-adenosine sequence.
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122. A method of analyzing the nucleic acid of a plurality of cells comprising:
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providing a plurality of partitions, wherein each partition comprises; a particle comprising a population of oligonucleotides having a barcode unique for that particle and a capture sequence, wherein the oligonucleotides comprise a 5′
end that is linked to the particle and a 3′
end that is available for ligation and/or extension by a polymerase; anda sample comprising a target nucleic acid; optionally cleaving the oligonucleotides conjugated to the plurality of particles from the particles; hybridizing the capture sequence, or a portion thereof, of the oligonucleotides to at least a portion of the target nucleic acid in each partition; performing template directed nucleic acid polymerization of the hybridized oligonucleotides, thereby covalently attaching the oligonucleotides to at least a portion of the target nucleic acid in each partition, wherein the template directed nucleic acid polymerization is performed before or after combining partitions; combining the partitions; and performing high throughput sequencing. - View Dependent Claims (123, 134, 135)
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- 124. The method of claim 122, wherein the method comprises performing template directed nucleic acid polymerization of the hybridized oligonucleotides, and then combining the partitions.
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124-1. (canceled)
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127. A method of analyzing the nucleic acid of a plurality of cells comprising:
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providing a plurality of partitions, wherein each partition comprises; a particle comprising a population of oligonucleotides having a barcode unique for that particle and a capture sequence, wherein the oligonucleotides comprise a 5′
end that is linked to the particle and a 3′
end that is available for ligation and/or extension by a polymerase; anda sample comprising a target nucleic acid; optionally cleaving the oligonucleotide primers conjugated to the plurality of particles from the particles; ligating the oligonucleotide primers to at least a portion of the target nucleic acid in each partition, thereby covalently attaching the oligonucleotides to at least a portion of the target nucleic acid in each partition, wherein the ligating is performed before combining partitions; combining the partitions; and performing high throughput sequencing. - View Dependent Claims (128, 129, 130, 131, 132)
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Specification