Specific double-stranded probes for homogeneous detection of nucleic acid and their application methods

• US 20040023269A1
• Filed: 08/12/2003
• Published: 02/05/2004
• Est. Priority Date: 10/10/2000
• Status: Active Grant

• Current Assignee: QuanDx, Inc.
• Original Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: Li, Qingge, Luan, Guoyan, Liang, Jixuan
• Granted Patent: US 7,799,522 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

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.

Claims

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.

2. A double-stranded probe according to claim 1 wherein said first and second oligonucleotides hybridize to produce a double-stranded blunt end, and wherein said fluorophore label and said second label are attached to said blunt end.

3. A double-stranded probe according to either of claim 1 or claim 2, wherein said first and second oligonucleotides have 3′

ends that are blocked from being extendable by a polymerase.

4. A double-stranded probe according to any of claims 1-3 wherein at least one of said first and second oligonucleotides comprises at least one non-natural nucleotide or at least one non-natural nucleotide linkage.

15. An assay for detecting a nucleic acid target sequence comprising adding to a sample suspect to contain the target sequence a probe according to any of claims 1-4 and measuring a change in fluorescence.

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.

6. A real-time amplification reaction according to claim 5, wherein said sequence first and second oligonucleotides hybridize to produce a double-stranded blunt end, and wherein said fluorophore label and said second label are attached to said blunt end.

7. A real-time amplification reaction according to either of claim 5 or claim 6, wherein said first and second oligonucleotides have 3′

ends that are blocked from being extendable by a polymerase.

8. A real-time amplification reaction according to any of claims 5-7 wherein at least one of said first and second oligonucleotides comprises at least one non-natural nucleotide or at least one non-natural nucleotide linkage.

9. A real-time amplification reaction according to any of claims 5-8 wherein said second sequence is shorter than said first sequence by up to ten nucleotides.

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.

11. Real-time amplification reaction according to claim 10 wherein the second sequence of each double-stranded probe is shorter than the first sequence of that probe by up to ten nucleotides.

12. A real-time amplification reaction according to either of claim 10 or claim 11, wherein the first and second nucleotides of each probe hybridize to produce a double-stranded blunt end, and wherein the fluorophore and quencher labels are attached to said blunt end.

13. A real-time amplification reaction according to any of claims 10-12 wherein the first and second oligonucleotides of said probes have 3′

ends that are blocked from being extended by a polymerase.

14. A real-time amplification reaction according to any of claims 10-13 wherein at least one of the first and second oligonucleotides of at least one of said probes comprises at least one non-natural nucleotide or at least one non-natural nucleotide linkage.