US5866336A
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Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon
Assignee: EMD Millipore Corp
•
Filing: 1997-01-03
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Granted: 1999-02-02
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Expires: 2017-01-03
Abstract
Abstract The present invention provides labeled nucleic acid amplification oligonucleotides, which can be linear or hairpin primers or blocking oligonucleotides. The oligonucleotides of the invention are labeled with donor and/or acceptor moieties of molecular energy transfer pairs. The moieties can be fluorophores, such that fluorescent energy emitted by the donor is absorbed by the acceptor. The acceptor may be a fluorophore that fluoresces at a wavelength different from the donor moiety, or it may be a quencher. The oligonucleotides of the invention are configured so that a donor moiety and an acceptor moiety are incorporated into the amplification product. The invention also provides methods and kits for directly detecting amplification products employing the nucleic acid amplification primers. When labeled linear primers are used, treatment with exonuclease or by using specific temperature eliminates the need for separation of unincorporated primers. This "closed-tube" format greatly reduces the possibility of carryover contamination with amplification products, provides for high throughput of samples, and may be totally automated.
Inventors
- 👤 Irina A. Nazarenko
- 👤 Satish K. Bhatnagar
- 👤 Emily S. Winn-Deen
- 👤 Robert J. Hohman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 13:59
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686; C12Q1/6813; C12Q1/6816; C12Q1/6818
Claims Analysis
Total Claims: 42
Claim 1
Independent Claim
101 words
An oligonucleotide for use as a primer in detecting a target nucleotide sequence, said oligonucleotide comprising: (a) a first nucleotide sequence complementary to a sequence flanking said target sequence; (b) a second nucleotide sequence at the 5' end of said first sequence; (c) a third nucleotide sequence at the 5' end of said second sequence; (d) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and (e) means for emitting a detectable signal when the strands of said duplex are separated.
Claim 2
Dependent Claim
101 words
An oligonucleotide for use as a primer in detecting a target nucleotide sequence, said oligonucleotide comprising: (a) a first nucleotide sequence complementary to a sequence flanking said target sequence; (b) a second nucleotide sequence at the 5' end of said first sequence; (c) a third nucleotide sequence at the 5' end of said second sequence; (d) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and (e) means for emitting a detectable signal when the strands of said duplex are separated.
Claim 3
Dependent Claim
43 words
The oligonucleotide according to claim 1 wherein said signal emitting means comprises an energy donor moiety and an energy acceptor moiety, each bound to said oligonucleotide and spaced such that said signal is detectable only when the strands of said duplex are separated.
Claim 4
Dependent Claim
23 words
The oligonucleotide according to claim 2 wherein said energy donor moiety is a fluorophore and said energy acceptor moiety is a fluorophore quencher.
Claim 5
Dependent Claim
126 words
A method for the amplification and detection of a target nucleotide sequence in a sample comprising the steps of: (a) providing a pair of primers each complementary to said target nucleotide sequence, at least one member of said primer pair comprising the detecting oligonucleotide of claim 1; (b) separating the strands of the nucleic acid containing the target nucleotide sequence; (c) annealing said pair of primers to the opposite strands of said separated nucleic acid; (d) synthesizing new strands of nucleic acid complementary to the strands of said separated nucleic acid; (e) separating said new strands from their complementary strands; and (f) repeating steps (c)-(e) wherein the synthesis of new strands separates the duplex strands of said oligonucleotide, thereby causing said detectable signal to be emitted.
Claim 6
Dependent Claim
142 words
A kit for use in detecting a target nucleotide sequence comprising: (a) first and second oligonucleotide primers at least one of which comprises: (i) a 3' nucleotide sequence that is complementary to a sequence flanking said target nucleotide sequence; (ii) a 5' nucleotide sequence that is not complementary to a sequence flanking said target sequence; and (b) a third oligonucleotide primer comprising: (i) a first sequence identical to said 5' sequence; (ii) a second sequence at the 5' end of said first sequence; (iii) a third nucleotide sequence at the 5' end of said second sequence; (iv) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and (v) means for emitting a detectable signal when the strands of said duplex are separated.
Claim 7
Dependent Claim
142 words
A kit for use in detecting a target nucleotide sequence comprising: (a) first and second oligonucleotide primers at least one of which comprises: (i) a 3' nucleotide sequence that is complementary to a sequence flanking said target nucleotide sequence; (ii) a 5' nucleotide sequence that is not complementary to a sequence flanking said target sequence; and (b) a third oligonucleotide primer comprising: (i) a first sequence identical to said 5' sequence; (ii) a second sequence at the 5' end of said first sequence; (iii) a third nucleotide sequence at the 5' end of said second sequence; (iv) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and (v) means for emitting a detectable signal when the strands of said duplex are separated.
Claim 8
Dependent Claim
17 words
The kit according to claim 5 wherein said 5' nucleotide sequence is not a naturally occurring sequence.
Claim 9
Dependent Claim
23 words
The oligonucleotide of claim 2 wherein said energy donor and acceptor moieties are spaced a distance in the range of about 10-40 nucleotides.
Claim 10
Dependent Claim
27 words
The oligonucleotide of claim 2 wherein said acceptor moiety is a fluorophore that emits fluorescent light at a wavelength different than that emitted by said donor moiety.
Claim 11
Dependent Claim
25 words
The oligonucleotide of claim 1 wherein said target nucleotide sequence is selected from the group consisting of genomic DNA, cDNA, mRNA, and chemically synthesized DNA.
Claim 12
Dependent Claim
18 words
The oligonucleotide of claim 1 wherein said target nucleotide sequence is a sequence of an infectious disease agent.
Claim 13
Dependent Claim
30 words
The oligonucleotide of claim 1 wherein said target nucleotide sequence is a wild-type human genomic sequence, mutation of which is implicated in the presence of a human disease or disorder.
Claim 14
Dependent Claim
59 words
The oligonucleotide of claim 2 wherein said donor moiety is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, and Reactive Red 4, and said acceptor moiety is selected from the group consisting of DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, fluorescein, Malachite green, and Texas Red.
Claim 15
Dependent Claim
21 words
The oligonucleotide of claim 12 wherein said donor moiety is fluorescein or a derivative thereof, and said acceptor moiety is DABCYL.
Claim 16
Dependent Claim
19 words
The oligonucleotide of claim 1 wherein said first or third nucleotide sequence further comprises a restriction endonuclease recognition site.
Claim 17
Dependent Claim
28 words
The oligonucleotide of claim 2 wherein said energy donor moiety and said energy acceptor moiety are situated on complementary nucleotides that are opposite each other in said duplex.
Claim 18
Dependent Claim
29 words
The oligonucleotide of claim 2 wherein said energy donor moiety and said energy acceptor moiety are situated on opposite strand nucleotides that are five nucleotides apart in said duplex.
Claim 19
Dependent Claim
57 words
A kit comprising in one or more containers: (a) a first oligonucleotide; and (b) a second oligonucleotide, wherein said first and second oligonucleotides are primers for use in a nucleic acid amplification reaction to amplify a preselected target nucleic acid sequence, and at least one of said first and second oligonucleotides is the oligonucleotide of claim 1.
Claim 20
Dependent Claim
27 words
The kit of claim 17 which further comprises a blocking oligonucleotide comprising a sequence complementary and hybridizable to a sequence of said first or said second oligonucleotide.
Claim 21
Dependent Claim
36 words
The kit of claim 17 which further comprises in one or more containers: (c) an optimized buffer for said amplification reaction; (d) a control nucleic acid comprising the preselected target sequence; and (e) a DNA polymerase.
Claim 22
Dependent Claim
133 words
A kit comprising in one or more containers: (a) a first oligonucleotide; (b) a second oligonucleotide, wherein said first and second oligonucleotides are primers for use in a nucleic acid amplification reaction to amplify a first preselected target nucleic acid sequence, and at least one of said first and second oligonucleotides is the oligonucleotide of claim 3; (c) a third oligonucleotide, and (d) a fourth oligonucleotide, wherein said third and fourth oligonucleotides are primers for use in said nucleic acid amplification reaction to amplify a second preselected target sequence, and at least one of said third and fourth oligonucleotides is an oligonucleotide of claim 3, and wherein said donor moiety of said first and second oligonucleotide emits fluorescent light of a different wavelength than said donor moiety of said third or fourth oligonucleotide.
Claim 23
Dependent Claim
25 words
The kit of claim 17 wherein said amplification reaction is selected from the group consisting of the polymerase chain reaction, strand displacement, triamplification and NASBA.
Claim 24
Dependent Claim
35 words
An oligodeoxynucleotide, the sequence of which consists of: 5'-ACCTTCTACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:13), wherein fluorescein or a derivative thereof is attached to the 5' A and DABCYL is attached to the T at nucleotide number 20.
Claim 25
Dependent Claim
35 words
An oligodeoxynucleotide, the sequence of which consists of: 5'-ACCTTCTACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:13), wherein fluorescein or a derivative thereof is attached to the 5' A and DABCYL is attached to the T at nucleotide number 20.
Claim 26
Dependent Claim
34 words
An oligodeoxynucleotide, the sequence of which consists: 5'-CACCTTCACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:18), wherein fluorescein or a derivative thereof is attached to the 5' C and DABCYL is attached to the T at nucleotide number 20.
Claim 27
Dependent Claim
34 words
An oligodeoxynucleotide, the sequence of which consists: 5'-CACCTTCACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:18), wherein fluorescein or a derivative thereof is attached to the 5' C and DABCYL is attached to the T at nucleotide number 20.
Claim 28
Dependent Claim
13 words
The kit of claim 17 wherein said first and second oligonucleotides are oligodeoxynucleotides.
Claim 29
Dependent Claim
100 words
A method for detecting or measuring a product of a nucleic acid amplification reaction comprising: (a) contacting a sample comprising nucleic acids with at least two oligonucleotide primers, said oligonucleotide primers being adapted for use in said amplification reaction such that said primers are incorporated into an amplified product of said amplification reaction when a preselected target sequence is present in the sample; at least one of said oligonucleotide primers being the oligonucleotide of claim 2; (b) conducting the amplification reaction; (c) stimulating energy emission from said donor moiety; and (d) detecting or measuring energy emitted by said acceptor moiety.
Claim 30
Dependent Claim
12 words
The method of claim 25 wherein said donor moiety is a fluorophore.
Claim 31
Dependent Claim
18 words
The method of claim 26 wherein said acceptor moiety is a quencher of light emitted by said fluorophore.
Claim 32
Dependent Claim
23 words
The method of claim 26 wherein said acceptor moiety emits fluorescent light of a wavelength different from that emitted by said donor moiety.
Claim 33
Dependent Claim
22 words
The method of claim 25 wherein said preselected target sequence is selected from the group consisting of genomic DNA, cDNA and mRNA.
Claim 34
Dependent Claim
58 words
The method of claim 25 wherein said donor moiety is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, and Reactive Red 4; and said acceptor moiety is selected from the group consisting DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, Malachite green, fluorescein and Texas Red.
Claim 35
Dependent Claim
21 words
The method of claim 25 wherein said donor moiety is fluorescein or a derivative thereof, and said acceptor moiety is DABCYL.
Claim 36
Dependent Claim
11 words
The method of claim 25 wherein the oligonucleotide is a oligodeoxynucleotide.
Claim 37
Dependent Claim
26 words
The method of claim 30 wherein said donor moiety and said acceptor moiety are situated on complementary nucleotides that are opposite each other in said duplex.
Claim 38
Dependent Claim
27 words
The method of claim 30 wherein said donor moiety and said acceptor moiety are situated on opposite strand nucleotides that are five nucleotides apart in said duplex.
Claim 39
Dependent Claim
94 words
The method of claim 30 wherein said oligonucleotide primers comprise a plurality of different oligonucleotides, each oligonucleotide comprising at its 3' end a said sequence complementary to different preselected target sequence whereby said different oligonucleotides are incorporated into different amplified products when each said target sequence is present in said sample, each said oligonucleotide being labeled with a donor moiety that emits light of a different wavelength than that emitted by the other donor moieties, and wherein step (d) of said method comprises detecting or measuring light emitted by each of the donor moieties.
Claim 40
Dependent Claim
28 words
The method of claim 30 wherein said amplification reaction is selected from the group consisting of polymerase chain reaction, allele-specific polymerase chain reaction, triamplification, strand displacement, and NASBA.
Claim 41
Dependent Claim
14 words
The kit of claim 17 which further comprises in a separate container DNA ligase.
Claim 42
Dependent Claim
32 words
The method of claim 25 which further comprises prior to said conducting step, contacting said nucleic acids with an amount of bisulfite sufficient to convert unmethylated cytosines in the sample to uracil.
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