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Microarray system with improved sequence specificity

  • US 8,067,164 B2
  • Filed: 08/12/2008
  • Issued: 11/29/2011
  • Est. Priority Date: 08/12/2007
  • Status: Expired due to Fees
First Claim
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1. A method of preparing a detectably labeled single-stranded polynucleotide target from a double stranded DNA comprising the detectably labeled polynucleotide target hybridized to a complementary polynucleotide comprising:

  • contacting the double stranded DNA with a 5′

    to 3′

    exonuclease under suitable conditions to degrade at least a portion of the complementary polynucleotide to form a detectably labeled single-stranded polynucleotide target, the polynucleotide target comprising no more than two phosphorothioate bonds and at least one of;

    (1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′

    end of the polynucleotide target;

    or(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′

    end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′

    end of the polynucleotide target;

    or(3) a cyanine dye moiety attached at the 5′

    end of the polynucleotide target, with the complementary strand modified with;

    (a) a 5′

    -phosphate group;

    or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

    -residue of said polynucleotide being an A or a T base;

    or(4) a dye moiety attached at the 5′

    end of the polynucleotide target and the first 5′

    -residue of said polynucleotide being a G or C base, with the complementary strand modified with;

    (a) a 5′

    -phosphate group;

    or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

    -residue of said polynucleotide being an A or a T base.

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