Single-labeled oligonucleotide probes for homogeneous nucleic acid sequence analysis

A probe for analyzing a target nucleic acid comprising

• a fluorescent detecting entity consisting essentially of a single-labeled oligonucleotide having a sequence generally complementary to a locus of the target nucleic acid and a fluorescent label linked to an internal residue of the oligonucleotide, the internal residue comprising a base analog, and
• wherein the sequence of the probe is selected so that upon hybridization of the probe to the locus of the target nucleic acid the magnitude of fluorescent emission from the fluorescent label is altered by hybridization of the probe to the target nucleic acid.

A method for determining the presence of a target nucleic acid sequence in a biological sample comprising:

• combining a single-labeled oligonucleotide probe with the sample, said probe having an oligonucleotide sequence generally complementary to a locus of the target nucleic acid sequence and a fluorescent label linked to a G residue of the oligonucleotide sequence, the fluorescent label exhibiting a hybridization-dependent fluorescent emission, wherein hybridization of the oligonucleotide probe to the target nucleic acid sequence alters interaction of the fluorescent label with the G residue, thereby increasing the fluorescent emission from the label,
• illuminating the biological sample, and
• monitoring the hybridization-dependent fluorescent emission.

A method of analyzing a sample comprising a target nucleic acid sequence, comprising the steps of

• combining the sample and an oligonucleotide probe to create a target-probe mixture, wherein the probe includes a virtual nucleotide having a fluorescent label positioned so that the magnitude of fluorescent emission from the fluorescent label is altered by hybridization of the probe to the target nucleic acid sequence,
• illuminating the mixture, and
• monitoring the fluorescent emission from the fluorescent label.

A kit for analyzing a biological sample comprising a nucleic acid sequence, comprising:

• a. a fluorescent detecting entity consisting essentially of a single-labeled oligonucleotide probe having an oligonucleotide comprising a base analog, the base analog linked to a fluorescent label, wherein said probe is configured to hybridize to a single-stranded locus of the segment so that the magnitude of fluorescent emission from the fluorescent label is increased by hybridization of the probe to the locus;
• and
• b. components for amplification of the nucleic acid sequence.