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1. 发明授权

US06821727B1 Hybridization assay using self-quenching fluorescence probe 失效
标题翻译：使用自熄荧光探针的杂交测定
公开(公告)号：US06821727B1
公开(公告)日：2004-11-23
申请号：US09627753
申请日：2000-07-28
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
IPC分类号： C12Q168
CPC分类号： C12Q1/6818 , C12Q1/6834 , Y10S977/924 , C12Q2525/301 , C12Q2565/1015 , C12Q2565/107 , C12Q2561/101
摘要： A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded confirmation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
摘要翻译：提供了使用寡核苷酸探针的杂交测定法，其包括荧光报告分子和能够猝灭报告分子荧光的猝灭剂分子。构建寡核苷酸探针，使得探针在未杂交的情况下以至少一种单链确认存在，其中淬灭剂分子足够接近报道分子以淬灭报道分子的荧光。当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。

2. 发明授权

US6030787A Hybridization assay using self-quenching fluorescence probe 有权
标题翻译：使用自熄荧光探针的杂交测定
公开(公告)号：US6030787A
公开(公告)日：2000-02-29
申请号：US207170
申请日：1998-12-07
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
IPC分类号： C12N15/09 , C07H21/00 , C12N15/00 , C12Q1/68 , C07H19/04 , C07H21/04
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837
摘要： A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
摘要翻译：提供了使用寡核苷酸探针的杂交测定法，其包括荧光报告分子和能够猝灭报告分子荧光的猝灭剂分子。构建寡核苷酸探针，使得探针在未杂交的情况下以至少一个单链构象存在，其中猝灭剂分子足够接近报道分子以淬灭报道分子的荧光。当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。

3. 发明授权

US5538848A Method for detecting nucleic acid amplification using self-quenching fluorescence probe 失效
标题翻译：使用自熄荧光探针检测核酸扩增的方法
公开(公告)号：US5538848A
公开(公告)日：1996-07-23
申请号：US340558
申请日：1994-11-16
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro
IPC分类号： C12N15/09 , C07H21/00 , C12N15/00 , C12Q1/68 , C07H21/04 , C12P19/34 , C12Q1/70
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837
摘要： A method is provided for monitoring the progress of nucleic acid amplifications that rely on a nucleic acid polymerase having 5'.fwdarw.3' exonuclease activity. An important feature of the method is providing an oligonucleotide probe having a reporter molecule and a quencher molecule at either end such that the quencher molecule substantially quenches any fluorescence from the reporter whenever the oligonucleotide probe is in a single stranded state and such that the reporter is substantially unquenched whenever the oligonucleotide probe is in a double stranded state hybridized to a target polynucleotide.
摘要翻译：提供了一种监测依赖于具有5'→3'核酸外切酶活性的核酸聚合酶的核酸扩增进程的方法。该方法的一个重要特征是提供了在任一端具有报告分子和猝灭剂分子的寡核苷酸探针，使得猝灭剂分子在寡核苷酸探针处于单链状态时基本上使来自报道分子的任何荧光猝灭，每当寡核苷酸探针处于与靶多核苷酸杂交的双链状态时，基本上未被消除。

4. 发明授权

US06258569B1 Hybridization assay using self-quenching fluorescence probe 有权
标题翻译：使用自熄荧光探针的杂交测定
公开(公告)号：US06258569B1
公开(公告)日：2001-07-10
申请号：US09436454
申请日：1999-11-08
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
IPC分类号： C12N100
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837 , C12Q2565/519 , C12Q2563/107 , C12Q2561/101 , C12Q2525/185 , C12Q2525/301 , C12Q2565/1015 , C12Q2565/107
摘要： Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.
摘要翻译：提供了核酸扩增的方法。在一个实施方案中，所述方法包括使用具有5'至3'核酸酶活性的核酸聚合酶在靶多核苷酸上进行核酸扩增，能够与靶多核苷酸杂交的引物和寡核苷酸探针在扩增条件下进行，使得探针与靶多核苷酸3'相对于引物杂交，并且探针不与其自身杂交以形成发夹结构。寡核苷酸探针在一端具有荧光报告基团，另一端是当荧光报道分子和猝灭剂连接到探针上时淬灭报道分子的荧光的猝灭剂。在扩增期间通过聚合酶消化寡核苷酸探针有效地将报道分子从猝灭剂中分离出来，从而增加了报道分子的荧光信号。

5. 发明授权

US5876930A Hybridization assay using self-quenching fluorescence probe 失效
公开(公告)号：US5876930A
公开(公告)日：1999-03-02
申请号：US558303
申请日：1995-11-15
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro , Khairuzzaman Bashar Mullah
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro , Khairuzzaman Bashar Mullah
IPC分类号： C12N15/09 , C07H21/00 , C12N15/00 , C12Q1/68 , C07H21/04
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837
摘要： A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

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