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1. 发明授权

US06610479B1 Activating a reversibly inactivated immobilized enzyme by release from an immobilizing moiety 失效
标题翻译：通过从固定部分释放来活化可逆灭活的固定化酶
公开(公告)号：US06610479B1
公开(公告)日：2003-08-26
申请号：US08952570
申请日：1998-02-13
申请人： Joakim Lundeberg , Mathias Uhlén
发明人： Joakim Lundeberg , Mathias Uhlén
IPC分类号： C12Q168
CPC分类号： C12Q1/6848 , C07K14/315 , C07K14/70535 , C07K2319/00 , C12N9/1252 , C12Q1/686 , Y10S435/81 , C12Q2549/107 , C12Q2527/127
摘要： Enzymes or catalytic fragments thereof reversibly inactivated by attachment to an immobilizing moiety which may comprise a magnetic particle are activated by release from the immobilizing moiety. The enzyme or fragment may be in the form of a fusion protein that is attached to the immobilizing moiety via a pair of affinity binding partners such that the enzyme or fragment is reversibly inactivated, and release of the fusion protein from the immobilizing moiety activates the enzyme or fragment. Enzymes include DNA polymerases, DNA ligases, Reverse transcriptases and RNA polymerases. The enzyme or fragment may be thermostable, and the fusion protein can be bound to the immobilizing moiety via a heat-labile linkage. Activation of the reversibly inactivated immobilized enzyme or fragment has particular utility in PCR and analogous nucleic acid amplification techniques. A sample containing a nucleic acid is contacted with the immobilized fusion protein, and release of the fusion protein activates the enzyme or fragment to start a first cycle of an amplification reaction. Amplification reactions that may be carried out include Ligase chain reaction (LCR), Self-sustained Sequence Replication (3SR), Reverse transcriptase PCR (RT-PCR), Q-beta replicase amplification reaction and nucleic acid sequence-based amplification (NASBA). Kits for the amplifications may be prepared containing an appropriate reversibly immobilized enzyme in the form of a fusion protein, and primers and/or probe for performing the amplification.
摘要翻译：通过与固定部分连接可逆地失活的酶或其催化片段可以由固定化部分释放而被活化。酶或片段可以是融合蛋白的形式，其通过一对亲和结合配偶体与固定化部分连接，使得酶或片段可逆地失活，并且融合蛋白从固定化部分的释放激活酶或片段。酶包括DNA聚合酶，DNA连接酶，逆转录酶和RNA聚合酶。酶或片段可以是热稳定的，并且融合蛋白可以通过热不稳定连接键与固定部分结合。可逆灭活的固定化酶或片段的活化在PCR和类似的核酸扩增技术中具有特别的用途。将含有核酸的样品与固定化的融合蛋白接触，并且融合蛋白的释放激活酶或片段以开始扩增反应的第一循环。可能进行的扩增反应包括连接酶链反应（LCR），自持序列复制（3SR），逆转录酶PCR（RT-PCR），Q-β复制酶扩增反应和基于核酸序列的扩增（NASBA）。可以制备用于扩增的试剂盒，其含有融合蛋白形式的合适的可逆固定的酶，以及用于进行扩增的引物和/或探针。

3. 发明授权

US06482592B1 Methods and kits for isolating primer extension products using modular oligonucleotides 失效
标题翻译：使用模块化寡核苷酸分离引物延伸产物的方法和试剂盒
公开(公告)号：US06482592B1
公开(公告)日：2002-11-19
申请号：US09153242
申请日：1998-09-15
申请人： Joakim Lundeberg , Mathias Uhlen
发明人： Joakim Lundeberg , Mathias Uhlen
IPC分类号： C12Q168
CPC分类号： C12Q1/707 , C12Q1/6832 , C12Q2537/162 , C12Q2525/185
摘要： The invention provides a method of improving the binding of a series of consecutive nucleotide bases to a complementary target nucleic acid molecule in a sample, wherein said method comprises at least the step or steps of binding a complementary modular oligonucleotide of at least two parts (modules) including said nucleotide bases to adjacent stretches of said target nucleic acid molecule in said sample, especially methods of detection/isolation, particularly in the isolation of primer extension products and methods in which the modular oligonucleotide is a primer, modular oligonucleotides themselves and their use in methods of the invention.
摘要翻译：本发明提供了一种改进一系列连续核苷酸碱基与样品中的互补靶核酸分子的结合的方法，其中所述方法包括至少两个部分的互补模块寡核苷酸（模块）结合的步骤或步骤）包括所述核苷酸碱基至所述样品中所述靶核酸分子的相邻延伸区，特别是检测/分离方法，特别是分离引物延伸产物和模块化寡核苷酸为引物的方法，模块化寡核苷酸本身及其用途在本发明的方法中。

4. 发明授权

US10030261B2 Method and product for localized or spatial detection of nucleic acid in a tissue sample 有权
公开(公告)号：US10030261B2
公开(公告)日：2018-07-24
申请号：US14111482
申请日：2012-04-13
申请人： Jonas Frisen , Patrik Ståhl , Joakim Lundeberg
发明人： Jonas Frisen , Patrik Ståhl , Joakim Lundeberg
IPC分类号： C12Q1/6837 , C12Q1/6834 , C40B20/02 , C12Q1/6841 , C12Q1/6844
摘要： The present invention relates to methods and products for the localized or spatial detection of nucleic acid in a tissue sample and in particular to a method for localized detection of nucleic acid in a tissue sample comprising: (a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′ end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′ to 3′: (i) a positional domain that corresponds to the position of the capture probe on the array, and (ii) a capture domain; (b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing nucleic acid of the tissue sample to hybridize to the capture domain in said capture probes; (c) generating DNA molecules from the captured nucleic acid molecules using said capture probes as extension or ligation primers, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain; (d) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying said tagged DNA; (e) releasing at least part of the tagged DNA molecules and/or their complements or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof; and (f) directly or indirectly analyzing the sequence of the released DNA molecules.

5. 发明授权

US5534424A Chemical method for the analysis of DNA sequences 失效
标题翻译：用于分析DNA序列的化学方法
公开(公告)号：US5534424A
公开(公告)日：1996-07-09
申请号：US331552
申请日：1995-04-18
申请人： Mathias Uhlen , Joakim Lundeberg
发明人： Mathias Uhlen , Joakim Lundeberg
IPC分类号： C12N15/09 , C12Q1/68 , C12P19/34 , C07H21/04 , C12Q1/70
CPC分类号： C12Q1/6858 , C12Q1/6834 , C12Q1/6869
摘要： The invention provides a method of identification of the base in a target position in a DNA sequence wherein sample DNA is subjected to amplification; the amplified DNA is immobilised and then subjected to strand separation, the non-immobilised strand being removed and an extension primer, which hybridises to the immobilised DNA immediately adjacent to the target position, is provided; each of four aliquots of the immobilised single stranded DNA is then subjected to a polymerase reaction in the presence of a dideoxynucleotide, each aliquot using a different dideoxynucleotide whereby only the dideoxynucleotide whereby only the dideoxynucleotide complementary to the base in the target position becomes incorpored; the four aliquots are then subjected to extension in the presence of all four deoxynucleotides, whereby in each aliquot the DNA which has not reacted with the dideoxynucleotide is extended to form double stranded DNA while the dideoxy-blocked DNA remains as non-extended DNA; followed by identification of the double stranded and/or non-extended DNA to indicate which dideoxynucleotide was incorporated and hence which base was present in the target position.
摘要翻译： PCT No.PCT / EP93 / 01203 371日期1995年04月18日 102（e）日期1995年4月18日PCT提交1993年5月12日PCT公布。公开号WO93 / 23562 日期：1993年11月25日。本发明提供了在DNA序列中的靶位置鉴定碱基的方法，其中对样品DNA进行扩增; 将扩增的DNA固定化，然后进行链分离，除去非固定化链和延伸引物，其与紧邻目标位置的固定化DNA杂交; 然后在双脱氧核苷酸存在的情况下，将四个等分的固定化单链DNA中的每一个进行聚合酶反应，每个等分试样使用不同的双脱氧核苷酸，从而只有双脱氧核苷酸，其中只有与目标位置的碱基互补的双脱氧核苷酸才会被吸收; 然后在四种脱氧核苷酸的存在下将四个等分试样延伸，由此在每个等分试样中，未与双脱氧核苷酸反应的DNA延伸形成双链DNA，而双脱氧封闭的DNA保留为非延伸的DNA; 随后鉴定双链和/或未延伸的DNA以指示哪个双脱氧核苷酸被引入，因此哪个碱基存在于目标位置。

6. 发明授权

US07262032B2 Allele-specific mutation detection assay using an enzyme-disabling agent 失效
标题翻译：使用酶灭活剂的等位基因特异性突变检测测定
公开(公告)号：US07262032B2
公开(公告)日：2007-08-28
申请号：US11256694
申请日：2005-10-21
申请人： Afshin Ahmadian , Joakim Lundeberg
发明人： Afshin Ahmadian , Joakim Lundeberg
IPC分类号： C12Q1/68 , C12P19/34 , C12N15/64 , C12N15/66 , C12N9/14
CPC分类号： C12Q1/6827 , C12Q1/6837 , C12Q1/6869 , C12Q2565/501 , C12Q2535/125 , C12Q2521/537 , C12Q2527/127 , C12Q2565/537
摘要： The present invention relates to a method of detecting a base at a pre-determined position in a nucleic acid molecule by performing enzymatic detection reactions using base-specific detection oligomers, where each oligomer is specific for a particular base at the predetermined position, and then comparing the enzymatic detection reactions to determine which base is present at the position, with an enzyme-disabling agent being present during the enzymatic detection reaction. In preferred embodiments the enzymatic detection reaction is an oligomer elongation extension reaction, catalysed by, among others, polymerase or ligase. Also disclosed are methods of performing the assay in a liquid phase and on microarrays.
摘要翻译：本发明涉及通过使用碱基特异性检测低聚物进行酶检测反应来检测核酸分子中预定位置的碱的方法，其中每个低聚物对于特定碱在预定位置是特异性的，然后比较酶检测反应以确定在该位置存在哪个碱基，在酶检测反应期间存在酶消失剂。在优选的实施方案中，酶检测反应是低聚物延伸延伸反应，其中包括聚合酶或连接酶催化。还公开了在液相和微阵列中进行测定的方法。

7. 发明申请

US20140066318A1 METHOD AND PRODUCT FOR LOCALIZED OR SPATIAL DETECTION OF NUCLEIC ACID IN A TISSUE SAMPLE 有权
标题翻译：在组织样品中局部或空间检测核酸的方法和产品
公开(公告)号：US20140066318A1
公开(公告)日：2014-03-06
申请号：US14111482
申请日：2012-03-13
申请人： Jonas Frisen , Patrik Ståhl , Joakim Lundeberg
发明人： Jonas Frisen , Patrik Ståhl , Joakim Lundeberg
IPC分类号： C12Q1/68
摘要： The present invention relates to methods and products for the localized or spatial detection of nucleic acid in a tissue sample and in particular to a method for localized detection of nucleic acid in a tissue sample comprising: (a) providing an array comprising a substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species occupies a distinct position on the array and is oriented to have a free 3′ end to enable said probe to function as a primer for a primer extension or ligation reaction, wherein each species of said capture probe comprises a nucleic acid molecule with 5′ to 3′: (i) a positional domain that corresponds to the position of the capture probe on the array, and (ii) a capture domain; (b) contacting said array with a tissue sample such that the position of a capture probe on the array may be correlated with a position in the tissue sample and allowing nucleic acid of the tissue sample to hybridize to the capture domain in said capture probes; (c) generating DNA molecules from the captured nucleic acid molecules using said capture probes as extension or ligation primers, wherein said extended or ligated DNA molecules are tagged by virtue of the positional domain; (d) optionally generating a complementary strand of said tagged DNA and/or optionally amplifying said tagged DNA; (e) releasing at least part of the tagged DNA molecules and/or their complements or amplicons from the surface of the array, wherein said part includes the positional domain or a complement thereof; and (f) directly or indirectly analyzing the sequence of the released DNA molecules.
摘要翻译：本发明涉及用于组织样本中核酸的局部或空间检测的方法和产品，特别涉及组织样品中核酸的局部检测方法，包括：（a）提供包含基质的阵列，直接或间接固定多种捕获探针，使得每个物种在阵列上占据不同的位置并且被定向为具有游离的3'末端以使得所述探针能够用作引物延伸或连接反应的引物，其中每个所述捕获探针的种类包括具有5'至3'的核酸分子：（i）对应于阵列上的捕获探针的位置的位置结构域，和（ii）捕获结构域; （b）使所述阵列与组织样品接触，使得阵列上的捕获探针的位置可与组织样品中的位置相关，并允许组织样品的核酸与所述捕获探针中的捕获结构域杂交; （c）使用所述捕获探针作为延伸或连接引物从捕获的核酸分子产生DNA分子，其中所述延伸或连接的DNA分子由于位置结构域而被标记; （d）任选地产生所述标记的DNA的互补链和/或任选地扩增所述标记的DNA; （e）从阵列的表面释放标记的DNA分子和/或其补体或扩增子的至少一部分，其中所述部分包括位置结构域或其互补体; 和（f）直接或间接分析释放的DNA分子的序列。

8. 发明申请

US20060088872A1 Allele-specific mutation detection assay 失效
标题翻译：等位基因特异性突变检测法
公开(公告)号：US20060088872A1
公开(公告)日：2006-04-27
申请号：US11256694
申请日：2005-10-21
申请人： Afshin Ahmadian , Joakim Lundeberg
发明人： Afshin Ahmadian , Joakim Lundeberg
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6827 , C12Q1/6837 , C12Q1/6869 , C12Q2565/501 , C12Q2535/125 , C12Q2521/537 , C12Q2527/127 , C12Q2565/537
摘要： The present invention relates to a method of detecting a base at a pre-determined position in a nucleic acid molecule by performing enzymatic detection reactions using base-specific detection oligomers, where each oligomer is specific for a particular base at the predetermined position, and then comparing the enzymatic detection reactions to determine which base is present at the position, with an enzyme-disabling agent being present during the enzymatic detection reaction. In preferred embodiments the enzymatic detection reaction is an oligomer elongation extension reaction, catalysed by, among others, polymerase or ligase. Also disclosed are methods of performing the assay in a liquid phase and on microarrays.
摘要翻译：本发明涉及通过使用碱基特异性检测低聚物进行酶检测反应来检测核酸分子中预定位置的碱的方法，其中每个低聚物对于特定碱在预定位置是特异性的，然后比较酶检测反应以确定在该位置存在哪个碱基，在酶检测反应期间存在酶消失剂。在优选的实施方案中，酶检测反应是低聚物延伸延伸反应，其中包括聚合酶或连接酶催化。还公开了在液相和微阵列中进行测定的方法。

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