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

• US 7,799,522 B2
• Filed: 10/05/2001
• Issued: 09/21/2010
• Est. Priority Date: 10/10/2000
• Status: Active Grant

• Current Assignee: QuanDx, Inc.
• Original Assignee: The Public Health Research Institute of the City of New York, Inc.
• Inventors: Li, Qingge, Luan, Guoyan, Liang, Jixuan
• Primary Examiner(s): Calamita; Heather
• Application Number:
  US10/398,832
• Publication Number:
  US 20040023269A1
• Time in Patent Office: 3,273 Days
• Field of Search: None
• US Class Current: 435/6.18
• 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 consisting of a first oligonucleotide consisting essentially of a first sequence 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 selected from the group consisting of a fluorescence 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, said probe being capable of spontaneously hybridizing to said target sequence in less than 30 minutes at 25°

C.

2. The 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. The double-stranded probe according to claim 2, wherein said first and second oligonucleotides have 3′

ends that are blocked from being extendable by a polymerase.

4. The double-stranded probe according to claim 1 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.

5. The double-stranded probe according to claim 1 wherein said second oligonucleotide is shorter than said first sequence by 1 to 5 nucleotides.

6. The double-stranded probe according to claim 1 wherein said second oligonucleotide is shorter than said first sequence by 2 to 7 nucleotides.

7. The double-stranded probe according to claim 1 wherein said first sequence is perfectly complementary to said target sequence.

8. The double-stranded probe according to claim 7 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.

9. A double-stranded nucleic acid hybridization probe for a preselected nucleic acid target sequence consisting of a first oligonucleotide consisting essentially of 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 selected from the group consisting of a fluorescence 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, wherein the free energy released by hybridization of the two oligonucleotides to one another is less than the free energy released by hybridization of the first oligonucleotide to the target sequence but greater than the free energy released by hybridization of the first oligonucleotide to a mismatched sequence that differs from the target sequence by a single nucleotide substitution.

10. The double-stranded probe according to claim 9 wherein said first and second oligonucleotides have 3′

ends that are blocked from being extendable by a polymerase.

11. The double-stranded probe according to claim 10 wherein said second oligonucleotide is shorter than said first sequence by 1-5 nucleotides.

12. The double-stranded probe according to claim 11 suitable for detecting said preselected target in a polymerase chain reaction (PCR) amplification reaction comprising primer extension, wherein said probe is melted off said target during said primer extension.

13. The double-stranded probe according to claim 10 wherein said second oligonucleotide is shorter than said first sequence by 2-7 nucleotides.