DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS
3 Assignments
0 Petitions
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.
-
Citations
337 Claims
-
1-307. -307. (canceled)
-
308. A method for determining a number of occurrences of a target molecule from a single cell, the method comprising:
-
a) generating an indexed library by performing a labeling step, the labeling step comprising; i) combining each of a target molecule from a plurality of target molecules from a single cell with a label-tag from a plurality of diverse label-tags, wherein the ratio of the number of the plurality of diverse label-tags to the number of the plurality of target molecules is greater than 5; and ii) generating the indexed library comprising a plurality of target-label-tag molecules by hybridizing the label-tag of the plurality of diverse label-tags to the target molecule of the plurality of target molecules and performing an extension reaction, wherein a target-label-tag molecule comprises the label-tag and a portion of a complementary sequence of the target molecule; b) amplifying the plurality of target-label-tag molecules from the indexed library to generate a plurality of amplified products; and c) sequencing at least a portion of the plurality of amplified products of step (b) to determine a number of different label-tag sequences associated with the portion of the complementary sequence of the target molecule from the single cell, wherein the number of different label-tag sequences indicates the number of occurrences of the target molecule from the single cell. - View Dependent Claims (309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337)
-
Specification