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公开(公告)号：US06379888B1
公开(公告)日：2002-04-30
申请号：US09406074
申请日：1999-09-27
IPC分类号： C12Q168
CPC分类号： C12Q1/6818 , Y10T436/143333 , C12Q2563/137 , C12Q2537/1373
摘要： Signal primers are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The signal primer comprises a first and a second oligonucleotide and is partially single-stranded and partially double-stranded. In the presence of target, the second oligonucleotide of the signal primer is displaced from the first and a conformational change in a reporter probe occurs which changes the distance between the members of a donor/quencher dye pair linked to the reporter probe. The change in proximity between the dyes causes an increase or a decrease in fluorescence quenching, which is detected as an indication of the presence of the target sequence.
摘要翻译： 信号引物用于通过荧光猝灭机制检测核酸靶序列。 信号引物包含第一和第二寡核苷酸并且是部分单链和部分双链的。 在靶的存在下，信号引物的第二寡核苷酸从第一个位点移位，并且发生报告物探针中的构象变化，其改变与报道探针连接的供体/猝灭剂染料对的成员之间的距离。 染料之间接近度的变化导致荧光淬灭的增加或减少，其被检测为靶序列存在的指示。
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公开(公告)号：US5919630A
公开(公告)日：1999-07-06
申请号：US186030
申请日：1998-11-04
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
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公开(公告)号：US5846726A
公开(公告)日：1998-12-08
申请号：US855085
申请日：1997-05-13
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
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公开(公告)号：US6054279A
公开(公告)日：2000-04-25
申请号：US120916
申请日：1998-07-20
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
摘要翻译： 通过连接到形成供体/受体染料对的两种染料来修饰单链信号引物。 这两种染料位于信号引物足够接近的空间上，第一种染料的荧光被第二种染料淬灭。 信号引物还可以包含两种染料之间的限制性内切核酸酶识别位点（RERS）。 由于信号引物最初是单链的，并且在不存在靶标的情况下保持单链，限制性内切核酸酶识别位点不受限制性内切核酸酶的切割或切割。 然而，在靶的存在下，信号引物和限制性内切核酸酶识别位点被限制性内切核酸酶双链和切割或切割。 切割或切口分离两种染料，并且由于猝灭降低引起的荧光变化被检测为目标序列或靶序列扩增的存在的指示。
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公开(公告)号：US5935791A
公开(公告)日：1999-08-10
申请号：US933749
申请日：1997-09-23
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/542 , G01N33/566 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6818
摘要： Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.
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公开(公告)号：US5928869A
公开(公告)日：1999-07-27
申请号：US865675
申请日：1997-05-30
CPC分类号： C12Q1/6827 , C12Q1/6818 , C12Q1/6858
摘要： A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.
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公开(公告)号：US06261784B1
公开(公告)日：2001-07-17
申请号：US09599164
申请日：2000-06-22
IPC分类号： C12Q168
CPC分类号： C12Q1/6818 , C12Q2537/1373 , C12Q2531/143 , C12Q2531/119
摘要： Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.
摘要翻译： 检测器核酸用于通过荧光猝灭机制检测核酸靶序列。 检测器核酸包含至少两个寡核苷酸并且是部分单链和部分双链的。 供体/受体染料对的两种染料之一与第一寡核苷酸连接，另一种与第二寡核苷酸连接，使得当第一和第二寡核苷酸是碱基配对并且供体荧光淬灭时它们处于紧密的空间接近 。 单个第二寡核苷酸可以与第一寡核苷酸杂交，或者多个第二寡核苷酸可以与第一寡核苷酸和彼此杂交，形成包含多个供体/受体染料对的连接结构。 检测器寡核苷酸在不存在目标时保留其部分单链和部分双链构象。 然而，在靶存在下，检测器核酸的第二寡核苷酸与第一寡核苷酸完全或部分位移，从而增加供体和受体染料之间的距离并引起荧光的变化，其可被检测为 指示靶序列的存在。
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公开(公告)号：US6130047A
公开(公告)日：2000-10-10
申请号：US235583
申请日：1999-01-22
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/542 , G01N33/566 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6818
摘要： Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.
摘要翻译： 检测器核酸用于通过荧光猝灭机制检测核酸靶序列。 检测器核酸包含至少两个寡核苷酸并且是部分单链和部分双链的。 供体/受体染料对的两种染料之一与第一寡核苷酸连接，另一种与第二寡核苷酸连接，使得当第一和第二寡核苷酸是碱基配对并且供体荧光淬灭时它们处于紧密的空间接近 。 单个第二寡核苷酸可以与第一寡核苷酸杂交，或者多个第二寡核苷酸可以与第一寡核苷酸和彼此杂交，形成包含多个供体/受体染料对的连接结构。 检测器寡核苷酸在不存在目标时保留其部分单链和部分双链构象。 然而，在靶存在下，检测器核酸的第二寡核苷酸与第一寡核苷酸完全或部分位移，从而增加供体和受体染料之间的距离并引起荧光的变化，其可被检测为 指示靶序列的存在。
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公开(公告)号：US5958700A
公开(公告)日：1999-09-28
申请号：US237510
申请日：1999-01-26
CPC分类号： C12Q1/6827 , C12Q1/6818 , C12Q1/6858
摘要： A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.
摘要翻译： 描述了具有形成分子内碱基配对二级结构的序列的检测器寡核苷酸用于检测核酸靶序列和靶序列扩增。 通过连接到形成供体/受体染料对的两种染料进一步修饰检测器寡核苷酸。 两种染料位于检测器寡核苷酸上，使得它们在碱基配对的折叠的二级结构中处于紧密的空间接近处，由此引起供体荧光的猝灭。 检测器寡核苷酸可以任选地进一步包含限制性内切核酸酶识别位点（RERS），所述限制性内切核酸酶识别位点在碱基配对二级结构中部分或全部保持单链。 RERS侧面有两种染料。 在靶的存在下，碱基配对的二级结构被展开或线性化，增加供体和受体染料之间的距离并引起供体和/或受体的荧光变化。 如果存在RERS，则在靶的存在下使其成为双链，允许通过限制性内切核酸酶切割或切割并将两种染料分离到单独的核酸片段上。 这可能进一步有助于荧光变化的大小。
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公开(公告)号：US5593867A
公开(公告)日：1997-01-14
申请号：US311474
申请日：1994-09-23
CPC分类号： C12Q1/6844
摘要： Fluorescence polarization methods for detection of nucleic acid amplification. A fluorescently labelled oligodeoxynucleotide probe is converted from single- to double-stranded form in a target dependent manner during amplification of the target sequence. This conformational change is accompanied by an increase in fluorescence polarization values. The increase in fluorescence polarization can be measured on a transient-state fluorometer in real-time during the amplification reaction without any physical manipulation of the sample. The inventive methods therefore provide a closed, homogeneous system for both amplification of target sequences and detection of amplification. Alternatively, amplification may be detected in the fluorometer after the amplification reaction is completed.
摘要翻译： 用于检测核酸扩增的荧光偏振方法。 荧光标记的寡脱氧核苷酸探针在靶序列扩增期间以靶依赖的方式从单链形式转化为双链形式。 这种构象变化伴随着荧光偏振值的增加。 可以在扩增反应期间在瞬态荧光计上实时测量荧光偏振的增加，而不对样品进行任何物理操作。 因此，本发明的方法提供用于靶序列扩增和扩增检测的封闭的均质系统。 或者，扩增反应完成后，可以在荧光计中检测扩增。
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