Molecular counting
First Claim
1. A method of determining a number of target molecules in a sample, the method comprising the steps of:
- (a) providing molecule-tag conjugates each comprising an oligonucleotide tag such that substantially every different molecule of the sample is attached to a different oligonucleotide tag, each oligonucleotide tag comprising a concatenation of subunits selected from a set of subunits, each subunit being a different nucleotide or oligonucleotide and having a position, and the set of subunits having a size of from 2 to 6 members;
(b) dividing the oligonucleotide tags of the molecule-tag conjugates into aliquots by sorting the oligonucleotide tags according to the identity of a subunit within a first or a successive position; and
(c) repeating step (b) for at least one aliquot in each successive application of step (b) until at least one aliquot has no oligonucleotide tags that can be separated into aliquots and determining the number of molecules in the sample from the number of times step (b) has been applied.
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Accused Products
Abstract
The invention provides methods and compositions for counting molecules in a sample, wherein each molecule is labeled with a unique oligonucleotide tag. Such tags are amplified and identified rather than the molecules themselves; that is, the problem of counting molecules is converted into the problem of counting tags. In one aspect of the invention, molecules to be counted are labeled by sampling. That is, conjugates are formed between the molecules to be counted and oligonucleotide tags of a very large set, or repertoire.
After conjugation, a sample of conjugates is taken that is sufficiently small so that substantially every molecule has a unique oligonucleotide tag. Counting of different tags may be accomplished in a variety of ways. In one aspect, different tags may be counted by carrying out a series of sorting steps to generate successively less complex mixtures in which tags are enumerated using length-encoded “metric” tags. In another aspect, different tags may be counted by directly sequencing a sample of tags using any one of several different sequencing methodologies.
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Citations
20 Claims
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1. A method of determining a number of target molecules in a sample, the method comprising the steps of:
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(a) providing molecule-tag conjugates each comprising an oligonucleotide tag such that substantially every different molecule of the sample is attached to a different oligonucleotide tag, each oligonucleotide tag comprising a concatenation of subunits selected from a set of subunits, each subunit being a different nucleotide or oligonucleotide and having a position, and the set of subunits having a size of from 2 to 6 members; (b) dividing the oligonucleotide tags of the molecule-tag conjugates into aliquots by sorting the oligonucleotide tags according to the identity of a subunit within a first or a successive position; and (c) repeating step (b) for at least one aliquot in each successive application of step (b) until at least one aliquot has no oligonucleotide tags that can be separated into aliquots and determining the number of molecules in the sample from the number of times step (b) has been applied. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of counting a number of target polynucleotides in a specimen, the method comprising the steps of:
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labeling by sampling each target polynucleotide in the specimen so that substantially every target polynucleotide is associated with a unique oligonucleotide tag, each oligonucleotide tag comprising a sorting tag and an identification tag; successively sorting the oligonucleotide tags a number of times by their sorting tags to form one or more separate mixtures; and determining the number of different oligonucleotide tags in at least one of the one or more separate mixtures by the identification tags thereof; and determining the number of target polynucleotides from the number of successive sortings and the number of different oligonucleotide tags in the one or more separate mixtures. - View Dependent Claims (9, 10, 11)
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12. A method of determining a number of target polynucleotides, the method comprising the steps of:
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providing for each target polynucleotide a plurality of nucleic acid probes specific for the target polynucleotide, each nucleic acid probe having a different oligonucleotide tag; combining in a reaction mixture the plurality of nucleic acid probes with the target polynucleotides so that substantially every target polynucleotide associates with a nucleic acid probe to form a selected nucleic acid probe that is resistant to a nuclease activity, the plurality of nucleic acid probes having a size sufficiently greater than the number of target polynucleotides so that substantially every selected nucleic acid probe has a unique oligonucleotide tag; isolating the selected nucleic acid probes by treating the reaction mixture with a nuclease having the nuclease activity; and determining nucleotide sequences of oligonucleotide tags in a sample of isolated selected nucleic acid probes to determine the number of different oligonucleotide tags therein, thereby determining the number of target polynucleotide in the mixture. - View Dependent Claims (13, 14, 15, 16)
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17. A method of estimating a number of target polynucleotides in a mixture, the method comprising the steps of:
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labeling by sampling each target polynucleotide in the mixture so that substantially every target polynucleotide has a unique oligonucleotide tag; amplifying the oligonucleotide tags of the labeled target polynucleotides; and determining the number of different oligonucleotide tags in a sample of amplified oligonucleotide tags by determining nucleotide sequences thereof, thereby estimating the number of target polynucleotides in the mixture. - View Dependent Claims (18, 19)
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20. A method of estimating a number of target polynucleotides in a mixture, the method comprising the steps of:
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labeling by sampling each target polynucleotide in the mixture so that substantially every target polynucleotide has a unique metric tag; amplifying the metric tags of the labeled target polynucleotides; separating the amplified metric tags to form a separation profile of distinct bands; and counting the number of distinct bands of metric tags, thereby estimating the number of target polynucleotides in the mixture.
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Specification