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Monitoring health and disease status using clonotype profiles

  • US 9,416,420 B2
  • Filed: 11/08/2013
  • Issued: 08/16/2016
  • Est. Priority Date: 11/07/2008
  • Status: Active Grant
First Claim
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1. A method of simultaneously measuring lymphocyte numbers and cellular clonotype expression levels in a sample, the method comprising the steps of:

  • extracting RNA from a sample comprising T cells and/or B cells;

    reverse transcribing the extracted RNA to form recombined cDNA molecules capable of encoding immune receptor molecules;

    extracting genomic DNA comprising recombined DNA molecules capable of encoding immune receptor molecules from the sample and adding a known quantity of an exogenous internal standard to the extracted genomic DNA,spatially isolating individual molecules of the exogenous internal standard and recombined DNA molecules from the extracted genomic DNA on a solid support;

    sequencing spatially isolated individual molecules of the exogenous internal standard and recombined DNA molecules from the extracted genomic DNA to determine a number of lymphocytes in the sample by comparing the number of sequence reads of the exogenous internal standard and the number of sequence reads of recombined DNA molecules from the genomic DNA;

    spatially isolating individual recombined cDNA molecules on a solid support;

    sequencing spatially isolated individual recombined cDNA molecules to obtain sequence reads corresponding to cellular expression levels of clonotypes for lymphocytes of the sample; and

    determining cellular clonotype expression levels in lymphocytes of the sample by comparing the number of sequence reads determined from the spatially isolated individual recombined DNA molecules from the genomic DNA and the number of sequence reads determined from the spatially isolated individual recombined cDNA molecules from RNA for each clonotype.

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