US6596490B2
Expired / Lapsed
US
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Nucleic acid hairpin probes and uses thereof
Assignee: Applied Gene Technologies Inc
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Filing: 2001-03-30
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Granted: 2003-07-22
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Expires: 2021-03-30
Abstract
Abstract This invention relates generally to nucleic acid hybridization analysis. More specifically, an oligonucleotide probe for hybridization analysis is provided, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with. Arrays comprising the hairpin probes immobilized on a solid support and methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided. Methods for transcribing and/or amplifying a probe DNA sequence using a hairpin probe are further provided.
Inventors
- 👤 Nanibhushan Dattagupta
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 05:53
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/6816; C12Q1/6834; C12Q1/6837; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
285 words
A method for detecting a target nucleotide sequence in a sample, which method comprises the steps of: a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a stein region comprising a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region tat is complementary to a target nucleotide sequence to be detected; b) contacting said probe provided in step a) with a sample containing or suspected of containing said target nucleotide sequence under conditions that favor intermolecular hybridization between said probe and said target nucleotide sequence over intramolecular hybridization of said probe itself, whereby said contacting breaks said intramolecular hybridization of said probe itself by dissociating said double stranded segment of said probe and forms a first intermolecular hybrid between said target and a region consisting of at least a portion of one stand of said stem and at least a portion of said loop within said probe that is complementary to said target nucleotide sequence; and c) assessing said intermolecular hybrid formed in step b), wherein said assessing is effected by addition of a secondary probe tat specifically hybridizes with at least a portion of said intermolecular hybrid, thereby forming a secondary intermolecular hybrid formed between said secondary probe and said first intermolecular hybrid wherein said detection of said secondary intermolecular hybrid indicates the presence and/or amount of said target nucleotide sequence in said sample.
Claim 2
Independent Claim
285 words
A method for detecting a target nucleotide sequence in a sample, which method comprises the steps of: a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a stein region comprising a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region tat is complementary to a target nucleotide sequence to be detected; b) contacting said probe provided in step a) with a sample containing or suspected of containing said target nucleotide sequence under conditions that favor intermolecular hybridization between said probe and said target nucleotide sequence over intramolecular hybridization of said probe itself, whereby said contacting breaks said intramolecular hybridization of said probe itself by dissociating said double stranded segment of said probe and forms a first intermolecular hybrid between said target and a region consisting of at least a portion of one stand of said stem and at least a portion of said loop within said probe that is complementary to said target nucleotide sequence; and c) assessing said intermolecular hybrid formed in step b), wherein said assessing is effected by addition of a secondary probe tat specifically hybridizes with at least a portion of said intermolecular hybrid, thereby forming a secondary intermolecular hybrid formed between said secondary probe and said first intermolecular hybrid wherein said detection of said secondary intermolecular hybrid indicates the presence and/or amount of said target nucleotide sequence in said sample.
Claim 3
Dependent Claim
37 words
The method of claim 1 , wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is substantially complementary to its corresponding nucleotide sequence in the target nucleotide sequence.
Claim 4
Dependent Claim
38 words
The method of claim 1 , wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is a perfect match to its corresponding nucleotide sequence in the target nucleotide sequence.
Claim 5
Dependent Claim
28 words
The method of claim 1 , wherein the single stranded loop of the probe comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or 15 nucleotides.
Claim 6
Dependent Claim
58 words
The method of claim 1 , wherein the conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself is achieved by controlling compositions of the probe and the target nucleotide sequence so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid.
Claim 7
Dependent Claim
26 words
The method of claim 5 , wherein the Tm of the intermolecular hybrid is at least 2° C. higher than the Tm of the intramolecular hybrid.
Claim 8
Dependent Claim
38 words
The method of claim 5 , wherein the intermolecular hybrid is a RNA:DNA, RNA:RNA or a DNA:DNA hybrid or a derivative thereof and the intramolecular hybrid is a RNA:DNA, RNA:RNA or a DNA:DNA hybrid or a derivative thereof.
Claim 9
Dependent Claim
22 words
The method of claim 5 , wherein the intermolecular hybrid is a RNA:DNA hybrid whereas the intramolecular hybrid is a DNA:DNA hybrid.
Claim 10
Dependent Claim
14 words
The method of claim 1 , wherein the second probe has a detectable label.
Claim 11
Dependent Claim
30 words
The method of claim 9 , wherein the label is selected from the group consisting of a chemical, an enzymatic, a radioactive, a fluorescent, a luminescent and a FRET label.
Claim 12
Dependent Claim
43 words
The method of claim 1 , further comprising a step of crosslinking the stern region of the probe that has not been hybridized wit the target after the formation of the intermolecular hybrid but before the addition of the detectably labeled secondary probe.
Claim 13
Dependent Claim
29 words
The method of claim 11 , wherein the crosslinking step is effected via addition of a crosslinking agent subsequent to hybridization of the probe with the target nucleotide sequence.
Claim 14
Dependent Claim
28 words
The method of claim 1 , wherein the hairpin probe further comprises an element or a modification that facilitates intramolecular crosslinking of the probe upon a suitable treatment.
Claim 15
Dependent Claim
21 words
The method of claim 13 , wherein the element is a crosslinking agent that is activated by chemical or photoactive treatment.
Claim 16
Dependent Claim
35 words
The method of claim 1 , wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is not involved in the hybridization of the target nucleotide sequence and the probe.
Claim 17
Dependent Claim
62 words
The method of claim 1 , wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is involved in the hybridization of the target nucleotide sequence and the probe and the nucleotide sequence in the probe that is complementary to said portion of the target nucleotide sequence is removed concurrently with the addition of the secondary probe.
Claim 18
Dependent Claim
62 words
The method of claim 16 ,wherein the hairpin structure in the probe is formed between a DNA strand that contains methylphosphonates and a RNA strand that is complementary to the target nucleotide sequence and wherein the RNA strand, after forming a hybrid with the target nucleotide sequence but before the addition of the secondary probe, is removed by a RNase H treatment.
Claim 19
Dependent Claim
65 words
The method of claim 1 , wherein the secondary probe specifically hybridizes with a portion of the probe that is involved in the formation of the intramolecular hybrid but is not involved in the formation of the first intermolecular hybrid with the target nucleotide sequence and wherein said portion of the probe dissociates as a single strand after the formation of the first intermolecular hybrid.
Claim 20
Dependent Claim
14 words
The method of claim 1 , wherein the secondary probe is a specific probe.
Claim 21
Dependent Claim
14 words
(Amended) The method of claim 1 , wherein the probe is a degenerate probe.
Claim 22
Dependent Claim
15 words
The method of claim 1 , wherein the probe is immobilized on a solid support.
Claim 23
Dependent Claim
19 words
The method of claim 1 , wherein a plurality of the probes immobilized on a solid support is used.
Claim 24
Dependent Claim
12 words
The method of claim 1 , wherein the sample is a biosample.
Claim 25
Dependent Claim
14 words
The method of claim 1 , wherein a plurality of samples is assayed simultaneously.
Claim 26
Dependent Claim
28 words
The method of claim 1 , wherein the oligonucleotide probe and the target nucleotide sequence is contacted at a temperature from about 4° C. to about 90° C.
Claim 27
Dependent Claim
28 words
The method of claim 1 , wherein the oligonucleotide probe and the target nucleotide sequence is contacted for a time from about 1 minute to about 60 minutes.
Claim 28
Dependent Claim
28 words
The method of claim 1 , wherein the probe further comprises an element that is sensitive to nuclease digestion or a modification that is resistant to nuclease digestion.
Claim 29
Dependent Claim
16 words
The method of claim 27 , wherein the nuclease digestion is effected via a restriction enzyme.
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