Patent ID: 8067164

Claim:
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.