公开(公告)号：US5854033A

公开(公告)日：1998-12-29

申请号：US563912

申请日：1995-11-21

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12Q1/68 ,  C12P19/34 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12Q1/6851 ,  C12Q1/6804 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q1/6858 ,  C12Q1/6862 ,  C12Q1/6865

摘要： Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

公开(公告)号：US5312728A

公开(公告)日：1994-05-17

申请号：US878230

申请日：1992-05-04

申请人： Paul M. Lizardi ,  Fred R. Kramer ,  Sanjay Tyagi ,  Cesar E. Guerra ,  Hilda M. L. Buyoli ,  Barbara C. Chu ,  Gerald F. Joyce ,  Leslie E. Orgel

发明人： Paul M. Lizardi ,  Fred R. Kramer ,  Sanjay Tyagi ,  Cesar E. Guerra ,  Hilda M. L. Buyoli ,  Barbara C. Chu ,  Gerald F. Joyce ,  Leslie E. Orgel

IPC分类号： C12N15/09 ,  C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6867 ,  C12Q1/6813 ,  C12Q1/6816 ,  C12Q1/682 ,  Y10T436/143333

摘要： A probe for the detection of a nucleic acid target sequence containing a molecular switch comprising three essential elements: a probe sequence of 20-60 nucleotides surrounded by switch sequences of 10-40 nucleotides which are complementary to each other, wherein the state of the switch is useful for selectively generating a detectable signal if the probe is hybridized to a target; also, assays and kits utilizing such probes.

摘要翻译： 用于检测含有包含三个必需元件的分子开关的核酸靶序列的探针：由彼此互补的10-40个核苷酸的切换序列包围的20-60个核苷酸的探针序列，其中开关的状态 如果探针与目标杂交，则可用于选择性地产生可检测信号; 还有使用这种探针的测定和试剂盒。

公开(公告)号：US5059294A

公开(公告)日：1991-10-22

申请号：US77901

申请日：1987-11-16

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： B01D57/02 ,  C12Q1/68 ,  G01N27/447

CPC分类号： C12Q1/68 ,  B01D57/02 ,  C12Q1/6813 ,  G01N27/44773

摘要： An improved method and device for nucleic acid hybridization assay employing combined direct an alternating field electrophoresis are disclosed. In the method of the present invention, a sample is hybridized with nucleic acid probe and is contacted with a support medium where direct and alternating electric fields are applied. Under the influence of the electric fields, hybrid separates from non-specifically bound nucleic acid probe. The hybrid may be measured on the support medium itself as on a paper strip or in a cartridge containing support medium or may be blotted on an inert surface and then measured. The method an device are useful in the diagnosis of diseases. Kits are provided for assay of a large number of diseases.

摘要翻译： 公开了使用组合直接交变电场电泳的改进的用于核酸杂交测定的方法和装置。 在本发明的方法中，样品与核酸探针杂交，并与施加直接和交替电场的支持介质接触。 在电场的影响下，杂交与非特异性结合的核酸探针分离。 混合物可以在载体介质本身上测量，如在纸条上或在含有载体介质的筒中，或者可以在惰性表面上印迹，然后测量。 该装置可用于诊断疾病。 提供试剂盒用于大量疾病的检测。

公开(公告)号：US07618776B2

公开(公告)日：2009-11-17

申请号：US10896513

申请日：2004-07-22

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6851 ,  C12Q1/6804 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q1/6858 ,  C12Q1/6862 ,  C12Q1/6865 ,  C12Q2563/179 ,  C12Q2531/125 ,  C12Q2545/10 ,  C12Q2537/143 ,  C12Q2531/119 ,  C12Q2531/143 ,  C12Q2537/125 ,  C12Q2533/107 ,  C12Q2525/131 ,  C12Q2537/162 ,  C12Q2525/307

摘要： Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

公开(公告)号：US06824981B2

公开(公告)日：2004-11-30

申请号：US09929266

申请日：2001-08-13

申请人： Brian T. Chait ,  Darin R. Latimer ,  Paul M. Lizardi ,  Eric R. Kershnar ,  Jon S. Morrow ,  Matthew E. Roth ,  Martin J. Mattessich ,  Kevin J. McConnell

发明人： Brian T. Chait ,  Darin R. Latimer ,  Paul M. Lizardi ,  Eric R. Kershnar ,  Jon S. Morrow ,  Matthew E. Roth ,  Martin J. Mattessich ,  Kevin J. McConnell

IPC分类号： C12Q168

CPC分类号： G01N33/6842 ,  C07K1/047 ,  C07K1/1077 ,  C07K1/13 ,  C07K7/06 ,  C07K7/08 ,  G01N33/6803

摘要： Disclosed are compositions and methods for sensitive detection of one or multiple analytes. In general, the methods involve the use of special label components, referred to as reporter signals, that can be associated with, incorporated into, or otherwise linked to the analytes. In some embodiments, the reporter signals can be altered such that the altered forms of different reporter signals can be distinguished from each other. In some embodiments, sets of reporter signals can be used where two or more of the reporter signals in a set have one or more common properties that allow the reporter signals having the common property to be distinguished and/or separated from other molecules lacking the common property. In other embodiments, sets of reporter signal/analyte conjugates can be used where two or more of the reporter signal/analyte conjugates in a set have one or more common properties that allow the reporter signal/analyte conjugates having the common property to be distinguished and/or separated form other molecules lacking the common property. Reporter signals can also be in conjunction with analytes (such as in mixtures of reporter signals and analytes), where no significant physical association between the reporter signals and analytes occurs; or alone, where no analyte is present.

摘要翻译： 公开了用于敏感检测一种或多种分析物的组合物和方法。 通常，这些方法涉及使用称为报告信号的特殊标记成分，其可以与分析物相关联，并入或以其它方式连接到分析物上。 在一些实施方案中，可以改变报道信号，使得不同报道信号的改变形式可以彼此区分。 在一些实施方案中，可以使用一组报告信号，其中一组中的两个或更多个报告基因信号具有一个或多个共同性质，其允许具有共同性质的报道信号被区分和/或与其他分子不相同 属性。 在其它实施方案中，可以使用组的报告信号/分析物缀合物，其中组中的两个或多个报道信号/分析物缀合物具有一个或多个共同性质，其允许区分具有共同性质的报道信号/分析物缀合物， /或分离形成其他缺乏共同性质的分子。 记者信号也可以与分析物（例如报告信号和分析物的混合物）结合，其中报告信号和分析物之间不存在明显的物理结合; 或单独存在，其中不存在分析物。

公开(公告)号：US06797474B2

公开(公告)日：2004-09-28

申请号：US10038718

申请日：2002-01-02

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12Q168

CPC分类号： C12Q1/6851 ,  C12Q1/6804 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q1/6858 ,  C12Q1/6862 ,  C12Q1/6865 ,  C12Q2563/179 ,  C12Q2531/125 ,  C12Q2545/10 ,  C12Q2537/143 ,  C12Q2531/119 ,  C12Q2531/143 ,  C12Q2537/125 ,  C12Q2533/107 ,  C12Q2525/131 ,  C12Q2537/162 ,  C12Q2525/307

摘要： Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

公开(公告)号：US06677121B2

公开(公告)日：2004-01-13

申请号：US09855793

申请日：2001-05-15

申请人： Paul M. Lizardi ,  Matthew E. Roth ,  Li Feng ,  Cesar E. Guerra ,  Shane C. Weber ,  Joseph C. Kaufman ,  Darin R. Latimer

发明人： Paul M. Lizardi ,  Matthew E. Roth ,  Li Feng ,  Cesar E. Guerra ,  Shane C. Weber ,  Joseph C. Kaufman ,  Darin R. Latimer

IPC分类号： C12Q168

CPC分类号： C12Q1/6855 ,  C12Q1/6809 ,  C12Q1/6837 ,  Y10S977/924 ,  C12Q2525/191 ,  C12Q2525/179 ,  C12Q2521/313 ,  C12Q2565/513

摘要： Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method, referred to as Fixed Address Analysis of Sequence Tags (FAAST), involves generation of a set of nucleic acid fragments having a variety of sticky end sequences; indexing of the fragments into sets based on the sequence of sticky ends; associating a detector sequence with the fragments; sequence-based capture of the indexed fragments on a detector array; and detection of the fragment labels. Generation of the multiple sticky end sequences is accomplished by incubating the nucleic acid sample with one or more nucleic acid cleaving reagents. The indexed fragments are captured by hybridization and coupling, preferably by ligation, to a probe. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner. One form of the method allows determination of associations, in a nucleic acid molecule, of different combinations of known or potential sequences. Another form of the method assesses modification of sequences in nucleic acid molecules by basing cleavage of the molecules on the presence or absence of modification.

摘要翻译： 公开了用于综合分析核酸样品的方法和用于该方法的检测器组合物。 称为序​​列标签的固定地址分析（FAAST）的方法涉及产生具有各种粘性末端序列的一组核酸片段; 基于粘性末端的顺序将片段索引到集合中; 将检测器序列与片段相关联; 检测器阵列上索引片段的基于序列的捕获; 并检测片段标签。 通过将核酸样品与一种或多种核酸切割试剂孵育来实现多粘性末端序列的产生。 索引的片段通过杂交和偶联，优选通过连接被捕获到探针。 该方法允许复制的核酸样品以可再现和序列特异性方式快速且容易地编目。 该方法的一种形式允许确定核酸分子中已知或潜在序列的不同组合的缔合。 该方法的另一种形式通过在存在或不存在修饰的基础上分解裂解来评估核酸分子中序列的修饰。

公开(公告)号：US06632609B2

公开(公告)日：2003-10-14

申请号：US09841513

申请日：2001-04-24

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12Q168

CPC分类号： C12Q1/6862 ,  C12Q1/6804 ,  C12Q1/6809 ,  C12Q1/6816 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q2563/179 ,  C12Q2531/125 ,  C12Q2531/143 ,  C12Q2537/125 ,  C12Q2531/119 ,  C12Q2537/143 ,  C12Q2539/113 ,  C12Q2565/102 ,  C12Q2525/161 ,  C12Q2563/131 ,  C12Q2525/307

摘要： Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecules present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules. Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.

公开(公告)号：US06403319B1

公开(公告)日：2002-06-11

申请号：US09637384

申请日：2000-08-11

申请人： Paul M. Lizardi ,  Darin R. Latimer

发明人： Paul M. Lizardi ,  Darin R. Latimer

IPC分类号： C12Q168

CPC分类号： C12Q1/6853 ,  A61K38/00 ,  C07K14/70578 ,  C12Q1/6809 ,  C12Q1/6837 ,  Y10T436/143333 ,  C12Q2565/513 ,  C12Q2525/191 ,  C12Q2521/313

摘要： Disclosed is a method for the comprehensive analysis of nucleic acid samples and a detector composition for use in the method. The method involves amplifying nucleic acid fragments of interest using a primer that can form a hairpin structure; sequence-based coupling of the amplified fragments to detector probes; and detection of the coupled fragments. The amplified fragments are coupled by hybridization and coupling, preferably by ligation, to detector probes. A hairpin structure formed at the end of the amplified fragments facilitates coupling of the fragments to the probes. The method allows detection of the fragments where detection provides some sequence information for the fragments. The method allows a complex sample of nucleic acid to be quickly and easily cataloged in a reproducible and sequence-specific manner. The method can also be used to detect amplified fragments having a known sequence.

摘要翻译： 公开了用于综合分析核酸样品的方法和用于该方法的检测器组合物。 该方法包括使用可形成发夹结构的引物扩增感兴趣的核酸片段; 扩增片段与检测器探针的基于序列的偶联; 并检测耦合片段。 通过杂交和偶联，优选通过连接将扩增的片段偶联到检测器探针。 在扩增片段末端形成的发夹结构有助于片段与探针的偶联。 该方法允许检测片段，其中检测提供片段的一些序列信息。 该方法允许复制的核酸样品以可再现和序列特异性方式快速且容易地编目。 该方法也可用于检测具有已知序列的扩增片段。

公开(公告)号：US06329150B1

公开(公告)日：2001-12-11

申请号：US09602428

申请日：2000-06-23

申请人： Paul M. Lizardi ,  Michael Caplan

发明人： Paul M. Lizardi ,  Michael Caplan

IPC分类号： C12Q168

CPC分类号： C12Q1/6862 ,  C12Q1/6804 ,  C12Q1/6809 ,  C12Q1/6816 ,  C12Q1/6844 ,  C12Q1/6853 ,  C12Q2563/179 ,  C12Q2531/125 ,  C12Q2531/143 ,  C12Q2537/125 ,  C12Q2531/119 ,  C12Q2537/143 ,  C12Q2539/113 ,  C12Q2565/102 ,  C12Q2525/161 ,  C12Q2563/131 ,  C12Q2525/307

摘要： Disclosed are compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication. The method is useful for simultaneously detecting multiple specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of an association operation, an amplification operation, and a detection operation. The association operation involves association of one or more specially designed probe molecules, either wholly or partly nucleic acid, to target molecules of interest. This operation associates the probe molecules to a target molecules present in a sample. The amplification operation is rolling circle replication of circular nucleic acid molecules, termed amplification target circles, that are either a part of, or hybridized to, the probe molecules. A single round of amplification using rolling circle replication results in a large amplification of the amplification target circles. Following rolling circle replication, the amplified sequences are detected using combinatorial multicolor coding probes that allow separate, simultaneous, and quantitative detection of multiple different amplified target circles representing multiple different target molecules. Since the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that a large number of distinct target molecules can be detected simultaneously, and that differences in the amounts of the various target molecules in a sample can be accurately quantified. It is also advantageous that the DNA replication step is isothermal, and that signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme.