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Methods for identifying nucleic acid sequences

  • US 9,556,473 B2
  • Filed: 08/22/2013
  • Issued: 01/31/2017
  • Est. Priority Date: 02/15/2011
  • Status: Active Grant
First Claim
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1. A method for evaluating a nucleic acid sequence in a tissue section comprising:

  • incubating an RNA containing the nucleic acid sequence in the tissue section with a reverse transcriptase and a reverse transcription primer that is complementary to the RNA to generate cDNA, wherein the reverse transcription primer is modified so as to be capable of immobilization in the cells of the tissue section;

    digesting all or part of the RNA;

    hybridizing one or more padlock probes to the cDNA, wherein the padlock probe(s) comprise one or two terminal regions having the nucleic acid sequence;

    incubating the hybridized padlock probe(s) and cDNA with ligase under conditions to ligate the ends of the padlock probe(s);

    incubating the padlock probe(s) with a polymerase to create an amplified rolling circle amplification product; and

    ,sequencing the amplified rolling circle amplification product.

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