DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS
First Claim
1. A method, comprising:
- (a) attaching to one or more occurrences of a first target molecule and a second target molecule in a sample (i) a first label sequence randomly selected from a first set of label sequences and (ii) a second label sequence randomly selected from a second set of label sequences, thereby generating for each occurrence of the first target molecule and the second target molecule a new first molecule and a new second molecule, respectively,wherein each new first molecule and new second molecule comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a combination label sequence comprising a first label sequence and a second label sequence,wherein n1 and n2 are the numbers of the one or more occurrences of the first target molecule and the second target molecule, respectively, in the sample,wherein combinations of each of the first set of label sequences and each of the second set of label sequences comprises m different combination label sequences, andwherein the ratio of the greater of n1 and n2 to m is smaller than 0.2; and
(b) detecting the new first molecules and the new second molecules by detecting the combination label sequences present on the new first molecules and the new second molecules, wherein the detected new first molecules and the detected new second molecules indicate the number of the one or more occurrences of the first target molecule and the number of the one or more occurrences of the second target molecule, respectively.
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Accused Products
Abstract
Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.
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Citations
30 Claims
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1. A method, comprising:
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(a) attaching to one or more occurrences of a first target molecule and a second target molecule in a sample (i) a first label sequence randomly selected from a first set of label sequences and (ii) a second label sequence randomly selected from a second set of label sequences, thereby generating for each occurrence of the first target molecule and the second target molecule a new first molecule and a new second molecule, respectively, wherein each new first molecule and new second molecule comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a combination label sequence comprising a first label sequence and a second label sequence, wherein n1 and n2 are the numbers of the one or more occurrences of the first target molecule and the second target molecule, respectively, in the sample, wherein combinations of each of the first set of label sequences and each of the second set of label sequences comprises m different combination label sequences, and wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2; and (b) detecting the new first molecules and the new second molecules by detecting the combination label sequences present on the new first molecules and the new second molecules, wherein the detected new first molecules and the detected new second molecules indicate the number of the one or more occurrences of the first target molecule and the number of the one or more occurrences of the second target molecule, respectively. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification