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Digital counting of individual molecules by stochastic attachment of diverse labels

  • US 10,059,991 B2
  • Filed: 07/22/2016
  • Issued: 08/28/2018
  • Est. Priority Date: 12/15/2009
  • Status: Active Grant
First Claim
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1. A method for obtaining clonal populations derived from a first target molecule and a second target molecule in a sample comprising:

  • (a) attaching by a random manner to each of a plurality of occurrences of a first target molecule and a second target molecule a label from a set of diverse labels, wherein the set comprises labels with m different label sequences, thereby generating for each of the plurality of occurrences of the first target molecule and the second target molecule, a labeled first molecule and a labeled second molecule, respectively,wherein each of the labeled first molecule and the labeled second molecule comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a label sequence,wherein more than 90% of the labeled first molecules and the labeled second molecules have a label sequence that is different from the label sequences on the other labeled first molecules and the labeled second molecules, respectively,wherein n1 and n2 are numbers of occurrences of the first target molecule and the second target molecule, respectively, andwherein the ratio of the greater of n1 and n2 to m is smaller than 0.2;

    (b) amplifying the labeled first molecules and the labeled second molecules to create a plurality of first clonal populations and a plurality of second clonal populations, respectively, wherein a first clonal population of the plurality of first clonal populations comprises copies of the first target molecule with a first identical label sequence, and wherein a second clonal population of the plurality of second clonal populations comprises copies of the second target molecule with a second identical label sequence; and

    (c) detecting the label sequences present on the plurality of first clonal populations and the plurality of second clonal populations, thereby detecting the label sequences present on the labeled first molecules and the labeled second molecules.

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