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：US06383754B1

公开(公告)日：2002-05-07

申请号：US09637751

申请日：2000-08-11

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：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：US06261782B1

公开(公告)日：2001-07-17

申请号：US09544713

申请日：2000-04-06

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; 基于粘性末端的序列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：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：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：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：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.