Digital counting of individual molecules by stochastic attachment of diverse labels

  • US 9,845,502 B2
  • Filed: 12/18/2015
  • Issued: 12/19/2017
  • Est. Priority Date: 12/15/2009
  • Status: Active Grant
First Claim
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1. 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 each 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.

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