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.

公开(公告)号：US6143495A

公开(公告)日：2000-11-07

申请号：US754681

申请日：1996-11-21

申请人： Paul M. Lizardi ,  Michael Caplan

发明人： Paul M. Lizardi ,  Michael Caplan

IPC分类号： C12N15/09 ,  C12Q1/68 ,  C07H21/02 ,  C07H21/04 ,  C12P19/34

CPC分类号： C12Q1/6862 ,  C12Q1/6804 ,  C12Q1/6809 ,  C12Q1/6816 ,  C12Q1/6844 ,  C12Q1/6853

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：US06642034B2

公开(公告)日：2003-11-04

申请号：US09911226

申请日：2001-07-23

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12P1934

CPC分类号： C12Q1/6844 ,  C12Q2537/143 ,  C12Q2531/119 ,  C12Q2531/125 ,  C12Q2525/179

摘要： Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The method is based on stand displacement replication of the nucleic acid sequences of interest by multiple primers. In one preferred form of the method, referred to as multiple strand displacement amplification, two sets of primers are used, a right set and a left set. The primers in the right set are complementary to one strand of the nucleic acid molecule to be amplified and the primers in the left set are complementary to the opposite strand. The 5′ end of primers in both sets are distal to the nucleic acid sequence of interest when the primers have hybridized to the nucleic acid sequence molecule to be amplified. Amplification proceeds by replication initiated at each primer and continuing through the nucleic acid sequence of interest. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In another preferred form of the method, referred to as whole genome strand displacement amplification, a random set of primers is used to randomly prime a sample of genomic nucleic acid (or another sample of nucleic acid of high complexity). By choosing a set of primers which are sufficiently random, the primers in the set will be collectively, and randomly, complementary to nucleic acid sequences distributed throughout nucleic acid in the sample. Amplification proceeds by replication with a highly processive polymerase initiated at each primer and continuing until spontaneous termination. A key feature of this method is the displacement of intervening primers during replication by the polymerase. In this way, multiple overlapping copies of the entire genome to be synthesized in a short time.

公开(公告)号：US06316229B1

公开(公告)日：2001-11-13

申请号：US09357487

申请日：1999-07-20

申请人： Paul M. Lizardi ,  Xiaohua Huang

发明人： Paul M. Lizardi ,  Xiaohua Huang

IPC分类号： C12P1934

CPC分类号： C12Q1/682 ,  C12Q1/6827 ,  C12Q1/6837 ,  C12Q2525/307 ,  C12Q2531/125 ,  C12Q2565/501 ,  C12Q2533/107 ,  C12Q2561/125 ,  C12Q2565/537

摘要： Disclosed are compositions and a method for detecting single nucleic acid molecules using rolling circle amplification (RCA) of single-stranded circular templates, referred to as amplification target circles, primed by immobilized primers. In one form of the method, referred to as a bipartite primer rolling circle amplification, (BP-RCA), RCA of the amplification target circle (ATC) depends on the formation of a primer by target-mediated ligation. In the presence of a nucleic acid molecule having the target sequence, a probe and a combination probe/primer oligonucleotide can hybridize to adjacent sites on the target sequence allowing the probes to be ligated together. By attaching the first probe to a substrate such as a bead or glass slide, unligated probe/primer can be removed after ligation. The only primers remaining will be primers ligated, via the probe portion of the probe/primer, to the first probe. The ligated primer can then be used to prime replication of its cognate ATC. In this way, an ATC will only be replicated if the target sequence (to which its cognate probe/primer is complementary) is present. BP-RCA is useful, for example, for determining which target sequences are present in a nucleic acid sample, or for determining which samples contain a target sequence. In another form of the method, referred to as immobilized primer rolling circle amplification (IP-RCA), RCA of the ATC depends on incorporation of a target sequence in the ATC during its formation. If the target sequence has been incorporated, a primer that can hybridize to the sequence will prime RCA of the ATC. This form of the method is useful for determining which form or forms of a variable sequence are present in a nucleic acid sample.

摘要翻译： 公开了使用由固定化引物引发的称为扩增靶圆的单链圆形模板的滚环扩增（RCA）检测单个核酸分子的组合物和方法。 在该方法的一种形式中，称为双极引物滚环扩增（BP-RCA），扩增靶圆环（ATC）的RCA取决于通过靶介导的连接形成引物。 在具有靶序列的核酸分子的存在下，探针和组合探针/引物寡核苷酸可以与目标序列上的相邻位点杂交，使探针连接在一起。 通过将第一探针连接到诸如珠粒或载玻片的基底上，可以在连接后除去未结合的探针/引物。 剩下的唯一引物将是通过探针/引物的探针部分连接到第一探针的引物。 然后连接的引物可用于引发其同源ATC的复制。 以这种方式，如果目标序列（其同源探针/引物互补）存在，ATC将仅被复制。 例如，BP-RCA可用于确定核酸样品中存在哪些靶序列，或用于确定哪些样品含有靶序列。 在该方法的另一种形式中，称为固定化引物滚环扩增（IP-RCA），ATC的RCA取决于ATC在其形成过程中靶序列的结合。 如果靶序列已被并入，则可以与序列杂交的引物将引起ATC的RCA。 该形式的该方法可用于确定核酸样品中存在可变序列的哪种形式或形式。

公开(公告)号：US5925517A

公开(公告)日：1999-07-20

申请号：US439819

申请日：1995-05-12

申请人： Sanjay Tyagi ,  Fred R. Kramer ,  Paul M. Lizardi

发明人： Sanjay Tyagi ,  Fred R. Kramer ,  Paul M. Lizardi

IPC分类号： C12N15/09 ,  C07H21/00 ,  C07H21/04 ,  C12Q1/68 ,  C07H21/02

CPC分类号： C12Q1/6816 ,  C12Q1/6818 ,  C12Q1/6827 ,  C12Q1/6841

摘要： Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays.

摘要翻译： 用于检测核酸靶序列的单分子和双分子杂交探针包含靶补体序列，在不存在靶序列的情况下将探针保持在封闭构象中的亲和对，以及当探针处于封闭状态时相互作用的标记对 构象，或对于某些单分子探针，非交互式标记。 靶和补体序列的杂交将探针转移到开放构象。 由于标签对的相互作用减少或通过检测来自非交互式标签的信号，该位移是可检测的。 某些单分子探针可以区分不同于一个核苷酸的靶和非靶序列。 此外，通用的茎和试剂盒可用于构建所述探针。 此外，利用所述探针和试剂盒进行这种测定的测定。

公开(公告)号：US06773886B2

公开(公告)日：2004-08-10

申请号：US09994311

申请日：2001-11-26

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

发明人： Joseph C. Kaufman ,  Matthew E. Roth ,  Paul M. Lizardi ,  Li Feng ,  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, referred to as Binary Encoded Sequence Tags (BEST), involves generation of a set of nucleic acid fragments; adding an adaptor to the ends containing recognition site for cleavage at a site offset from the recognition site; cleaving the fragment to generate fragments having a plurality sticky ends; indexing of the fragments into sets based on the sequence of sticky ends. The fragments are indexed by adding a offset adaptor to newly generated ends. A different adaptor will be coupled to each different sticky end. The resulting fragments—which will have defined ends, be of equal lengths (in preferred embodiment), and a central sequence derived from the source nucleic acid molecule—are binary sequence tags. The binary sequence tags can be used and further analyzed in numerous ways. For example, the binary sequence tags can be captured by hybridization and coupling, preferably by ligation, to a probe. The probe is preferably immobilized in an array or on sortable beads. One form of the BEST method, referred to as modification assisted analysis of binary sequence tags (MAABST), assesses modification of sequences in nucleic acid molecules by detecting differential cleavage based on the presence or absence of modification in the molecules.

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。 称为二进制编码序列标签（BEST）的方法涉及产生一组核酸片段; 在包含识别位点的末端添加适配器以在与识别位点偏移的位点处切割; 切割片段以产生具有多个粘性末端的碎片; 基于粘性末端的顺序将片段索引到集合中。 通过向新生成的末尾添加偏移适配器来索引碎片。 不同的适配器将连接到每个不同的粘性端。 所得到的片段（其将具有定义的末端）具有相同的长度（在优选实施方案中），并且源自源核酸分子的中心序列是二进制序列标签。 二进制序列标签可以以多种方式使用和进一步分析。 例如，二元序列标签可以通过杂交和偶联（优选通过连接）到探针来捕获。 探针优选固定在阵列中或可排序的珠粒上。 BEST方法的一种形式，称为二元序列标签（MAABST）的修饰辅助分析，通过基于分子中存在或不存在修饰来检测差异切割来评估核酸分子中序列的修饰。

公开(公告)号：US06344329B1

公开(公告)日：2002-02-05

申请号：US09644723

申请日：2000-08-23

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：US06287824B1

公开(公告)日：2001-09-11

申请号：US09396281

申请日：1999-09-15

申请人： Paul M. Lizardi

发明人： Paul M. Lizardi

IPC分类号： C12P1934

CPC分类号： C12Q1/6809 ,  C12Q1/6827 ,  C12Q1/6844 ,  C12Q1/6853 ,  Y10T436/143333 ,  C12Q2565/501 ,  C12Q2537/137 ,  C12Q2523/101 ,  C12Q2563/131 ,  C12Q2531/125 ,  C12Q2527/143 ,  C12Q2565/537 ,  C12Q2525/131 ,  C12Q2531/119

摘要： Disclosed are reagents and a method for efficient in vitro molecular cloning of nucleic acid molecules of interest. Because the method is entirely in vitro, it can be automated and scaled-up in ways that are not possible in cell-based molecular cloning. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermic reaction that replicates the vector sequences exponentially. The amplification process is amenable to automation where multiple reactions are carried out simultaneously in a small area. The amplified nucleic acid can be used for any purpose and in any manner that nucleic acid cloned or amplified by known methods can be used. This includes sequencing, probing, restriction analysis, subcloning, transcription, hybridization or denaturation analysis, further amplified, and storage for future use or analysis.

摘要翻译： 公开了用于有效体外分子克隆感兴趣的核酸分子的试剂和方法。 因为这种方法是完全体外的，所以它可以通过基于细胞的分子克隆的方式自动化和放大。 该方法包括在线性载体中插入目的核酸分子以形成圆形载体，其中一条链是连续的，另一条链是不连续的。 然后通过滚环复制扩增圆形载体的连续链，在该过程中扩增插入的核酸分子。 扩增是快速和有效的，因为它涉及单一的等温反应，其以指数方式复制载体序列。 扩增过程适用于在小范围内同时进行多重反应的自动化。 扩增的核酸可以用于任何目的，也可以通过已知方法克隆或扩增的核酸的任何方式使用。 这包括测序，探测，限制性分析，亚克隆，转录，杂交或变性分析，进一步扩增和存储以供将来使用或分析。

公开(公告)号：US06183960B2

公开(公告)日：2001-02-06

申请号：US09132552

申请日：1998-08-11

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：US6103476A

公开(公告)日：2000-08-15

申请号：US268402

申请日：1999-03-15

申请人： Sanjay Tyagi ,  Fred R. Kramer ,  Paul M. Lizardi

发明人： Sanjay Tyagi ,  Fred R. Kramer ,  Paul M. Lizardi

IPC分类号： C12N15/09 ,  C07H21/00 ,  C07H21/04 ,  C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6816 ,  C12Q1/6818 ,  C12Q1/6827 ,  C12Q1/6841

摘要： Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays.

摘要翻译： 用于检测核酸靶序列的单分子和双分子杂交探针包含靶补体序列，在不存在靶序列的情况下将探针保持在封闭构象中的亲和对，以及当探针处于封闭状态时相互作用的标记对 构象，或对于某些单分子探针，非交互式标记。 靶和补体序列的杂交将探针转移到开放构象。 由于标签对的相互作用减少或通过检测来自非交互式标签的信号，该位移是可检测的。 某些单分子探针可以区分不同于一个核苷酸的靶和非靶序列。 此外，通用的茎和试剂盒可用于构建所述探针。 此外，利用所述探针和试剂盒进行这种测定的测定。