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LARGE-SCALE BIOMOLECULAR ANALYSIS WITH SSEQUENCE TAGS

  • US 20150259734A1
  • Filed: 06/27/2014
  • Published: 09/17/2015
  • Est. Priority Date: 07/01/2013
  • Status: Active Grant
First Claim
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1. A method of generating clonotype profiles from a plurality of Mutt le cell receptor chains, the method comprising the steps of:

  • (a) combining in a reaction mixture under primer extension conditions a first set of primers with a sample of recombined nucleic acids from B-cells and/or T-cells and/or cell-free DNA, wherein each printer of the first set has a receptor-specific portion such that the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined location and is extended to form a first extension product, and wherein each primer of the first set has a 5′

    non-eomplementary end containing a first primer binding site(b) removing from the reaction mixture non-extended primers of the first set;

    (c) adding to the reaction mixture under primer extension conditions a second set of primers, wherein each primer of the second set has a receptor-specific portion such that the receptor-specific portion anneals to the first extension product at a predetermined location and has a 5′

    -non-complementary end containing a second primer binding site, primers of the first set and/or primers of the second set comprising a sequence tag disposed between the receptor-specific portion and the first or second primer binding site, respectively, and wherein each primer of the second set is extended to form a second extension product, such that each second extension product comprises a first primer binding site, a second primer binding site, at least one sequence tag, and recombined nucleic acid encoding a portion or T cell receptor chain or a B cell receptor chain;

    (d) performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second primer binding site; and

    (e) sequencing the nucleic acids or the amplicon to form a clonotype profile of a plurality of T cell receptor chains and/or B cell receptor chains.

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