Specific double-stranded probes for homogeneous detection of nucleic acid and their application methods
First Claim
1. A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence comprising a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence, a second oligonucleotide comprising a second sequence that is complementary to said first sequence but is shorter than said first sequence by up to ten nucleotides, a fluorophore label attached to one of said first and second oligonucleotides, and a second label from the group consisting of a quencher and a fluorescence acceptor attached to the other of said first and second oligonucleotides so as to interact with said fluorophore label when said oligonucleotides are hybridized to each other.
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Abstract
Double-stranded nucleic acid hybridization probes comprise a longer strand perfectly complementary to a preselected target sequence in an assay and a shorter second strand complementary to the longer strand. The strands are labeled with interactive labels such as a fluorophore and a quencher. The probes may be used in real-time amplification assays to distinguish among alleles.
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Citations
15 Claims
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1. A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence comprising
a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence, a second oligonucleotide comprising a second sequence that is complementary to said first sequence but is shorter than said first sequence by up to ten nucleotides, a fluorophore label attached to one of said first and second oligonucleotides, and a second label from the group consisting of a quencher and a fluorescence acceptor attached to the other of said first and second oligonucleotides so as to interact with said fluorophore label when said oligonucleotides are hybridized to each other.
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5. In a real-time nucleic acid target sequence amplification and detection reaction in which target amplicons are detected by a fluorescently labeled hybridization probe, the improvement comprising using as said hybridization probe a double-stranded nucleic acid hybridization probe for said target sequence comprising
a first oligonucleotide comprising a first sequence perfectly complementary to said target sequence, a second oligonucleotide comprising a second sequence that is complementary to said first sequence, a fluorophore label attached to one of said first and second oligonucleotides, and a second label from the group consisting of a quencher and a fluorescence acceptor attached to the other of said first and second oligonucleotides so as to interact with said fluorophore label when said oligonucleotides are hybridized to each other.
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10. In real-time nucleic acid amplification and detection reaction in which at least two target sequences are alleles differing by at least a single nucleotide, and in which amplicons for each allelic target sequence are detected by a fluorescently labeled hybridization probe complementary thereto, the improvement wherein each hybridization probe is a double-stranded nucleic acid hybridization probe for its allelic target sequence comprising
a first oligonucleotide comprising a first sequence perfectly complementary to said allelic target sequence, a second oligonucleotide comprising a second sequence that is complementary to said first sequence, a fluorophore label attached to one of said first and second oligonucleotides, and a quencher label attached to the other of said first and second oligonucleotides so that the fluorophore and quencher are in a quenching relationship when said oligonucleotides are hybridized to each other, and the fluorophore label of each of said double-stranded probes is distinguishably detected.
Specification