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1. 发明授权

US10590453B2 Methods for amplification of nucleic acids utilizing a circularized template prepared from a target nucleic acid 有权
公开(公告)号：US10590453B2
公开(公告)日：2020-03-17
申请号：US15998679
申请日：2018-08-16
申请人： Lyle J. Arnold , Norman C. Nelson
发明人： Lyle J. Arnold , Norman C. Nelson
IPC分类号： C12P19/34 , C12Q1/6844
摘要： The present invention provides methods of amplifying a target nucleic acid utilizing a circularized template. Circularization may be achieved utilizing a bridging oligonucleotide or an inverter primer. The bridging oligonucleotide or inverted primer is extended forming a concatemeric amplicon that can then be used as a template to provide exponential amplification of the target nucleic acid.

2. 发明授权

US10174352B2 Methods for amplification of nucleic acids on solid support 有权
公开(公告)号：US10174352B2
公开(公告)日：2019-01-08
申请号：US14773362
申请日：2014-03-14
申请人： Lyle J. Arnold , Norman C. Nelson
发明人： Lyle J. Arnold , Norman C. Nelson
IPC分类号： C12Q1/68 , C12P19/34 , C12Q1/6853 , C12Q1/6865 , C07H21/02
摘要： The present invention provides methods for amplifying a nucleic acid from a sample containing a mixture of nucleic acids utilizing a solid support. Methods are provided utilizing user-defined primer oligonucleotides for directional amplification that assists in further manipulation of the target nucleic acid, such as sequencing. Methods are also provided utilizing blocker and displacer oligonucleotides for generating amplified target nucleic acids of defined length. One of these methods provides a first oligonucleotide and a second oligonucleotide affixed to a solid support or separate solid supports. The first oligonucleotide is blocked to prevent extension from the 3′-terminus and has a sequence complementary to a first portion of a target nucleic acid. The second oligonucleotide has a sequence that is identical to a second portion of the target nucleic acid. In this method, a sample is applied to the solid support and the target nucleic acid within the sample binds said first oligonucleotide. The solid support is then washed to remove unbound nucleic acids. A primer sequence containing a target binding region and a polymerase promoter sequence is then annealed to the bound target nucleic acid and extended producing a first duplex nucleic acid. The target sequence is then removed leaving a first nucleic acid that can now bind the second oligonucleotide. The second oligonucleotide is extended to produce a second duplex nucleic acid that contains a second nucleic acid. The second nucleic acid is then amplified by adding a polymerase.

3. 发明授权

US10081825B2 Methods for amplification of nucleic acids utilizing a circularized template prepared from a target nucleic acid 有权
公开(公告)号：US10081825B2
公开(公告)日：2018-09-25
申请号：US14773363
申请日：2014-03-14
申请人： Lyle J. Arnold , Norman C. Nelson
发明人： Lyle J. Arnold , Norman C. Nelson
IPC分类号： C12Q1/68 , C12P19/34 , C12Q1/6844
CPC分类号： C12P19/34 , C12Q1/6844 , C12Q2531/119 , C12Q2531/125 , C12Q2537/162
摘要： The present invention provides methods of amplifying a target nucleic acid utilizing a circularized template. Circularization may be achieved utilizing a bridging oligonucleotide or an inverter primer. The bridging oligonucleotide or inverted primer is extended forming a concatemeric amplicon that can then be used as a template to provide exponential amplification of the target nucleic acid.

4. 发明授权

US10072290B2 Methods for amplifying fragmented target nucleic acids utilizing an assembler sequence 有权
公开(公告)号：US10072290B2
公开(公告)日：2018-09-11
申请号：US14773366
申请日：2014-03-15
申请人： Lyle J. Arnold , Norman C. Nelson
发明人： Lyle J. Arnold , Norman C. Nelson
IPC分类号： C12Q1/68 , C12Q1/6858 , C12Q1/6806 , C12Q1/6844
CPC分类号： C12Q1/6858 , C12Q1/6806 , C12Q1/6844 , C12Q2525/161 , C12Q2525/186 , C12Q2525/204 , C12Q2531/101
摘要： The present invention provides methods of amplifying a fragmented target nucleic acid containing short target nucleic acid fragments utilizing an assembler sequence to convert these short fragments into longer sequences enabling their identification and interrogation. This is particularly important when attempting to identify small genetic variations, such as SNVs, present in highly fragmented nucleic acid samples. Amplification is accomplished by hybridizing the short target nucleic acid sequences to the assembler sequence, where these short sequences serve as primers for extension. Since the fragmented target nucleic acids that contain SNVs are utilized as primers on the assembler sequence they are preserved during amplification and can be detected.

5. 发明申请

US20160130623A1 Methods for Amplification of Nucleic Acids Utilizing Clamp Oligonucleotides 审中-公开
标题翻译：使用钳夹寡核苷酸扩增核酸的方法
公开(公告)号：US20160130623A1
公开(公告)日：2016-05-12
申请号：US14773365
申请日：2014-03-14
申请人： Lyle J. ARNOLD , Norman C. NELSON
发明人： Lyle J. Arnold
IPC分类号： C12P19/34
CPC分类号： C12P19/34 , C12Q1/6844 , C12Q2521/501 , C12Q2525/307 , C12Q2549/126
摘要： The present invention provides methods of amplifying a target nucleic acid utilizing a clamp oligonucleotide comprising a first target-binding region on the 3′-terminus and a second target-binding region on the 5′-terminus and tether region in between. The tether region may comprise a variety of user-defined sequences or elements that allow for further manipulation of the target nucleic acid. Such as, for example, capture followed by amplification, identification and/or sequencing. The target-binding regions bind to the target nucleic acid, the 3′-terminus functions as a primer to initiate extension across the target nucleic acid sequence and ligation of the gap results in formation of a circularized nucleic acid. This circular template can be used in a variety of processes, including amplification and sequencing.
摘要翻译：本发明提供了使用包含3'末端的第一靶结合区和5'末端之间的第二靶结合区和其间的系链区的钳位寡核苷酸来扩增靶核酸的方法。系链区域可以包含允许进一步操纵靶核酸的各种用户定义的序列或元件。例如，进行捕获，随后进行扩增，鉴定和/或测序。靶结合区域与靶核酸结合，3'-末端用作引物以引发靶核酸序列的延伸，并且间隙的连接导致形成环化的核酸。该圆形模板可用于各种过程，包括扩增和测序。

6. 发明申请

US20140274810A1 Methods for Amplification of Nucleic Acids Utilizing Hairpin Loop or Duplex Primers 有权
标题翻译：使用发夹环或双相引物扩增核酸的方法
公开(公告)号：US20140274810A1
公开(公告)日：2014-09-18
申请号：US14214621
申请日：2014-03-14
申请人： Lyle J. Arnold
发明人： Lyle J. Arnold
IPC分类号： C12N15/10 , C12Q1/68
CPC分类号： C12Q1/6853 , C12N15/1065 , C12Q2521/501 , C12Q2525/301 , C12Q2563/185
摘要： The present invention provides methods of amplifying a target nucleic acid utilizing duplex primer. The first strand of the primer comprises a random nucleotide sequence of about 6 to about 9 nucleotides in length that is able to hybridize to the target nucleic acid and a tag sequence. The second strand of the primer comprises a sequence complementary to the tag sequence allowing the primer to form a duplex and the ability to bind the tag sequence of the product nucleic acid for further amplification. The resulting nucleic acid produced contains tag sequences on both the 3′- and 5′-termini.
摘要翻译：本发明提供了利用双链体引物扩增靶核酸的方法。引物的第一条链包含长度为约6至约9个核苷酸的随机核苷酸序列，其能够与靶核酸和标签序列杂交。引物的第二条链包含与标签序列互补的序列，使得引物形成双链体并结合产物核酸的标签序列用于进一步扩增的能力。产生的所得核酸含有3'和5'末端的标签序列。

7. 发明申请

US20140274781A1 Methods for Immobilizing Target Nucleic Acids Utilizing Combinatorial Capture Probes 审中-公开
标题翻译：使用组合捕获探针固定目标核酸的方法
公开(公告)号：US20140274781A1
公开(公告)日：2014-09-18
申请号：US14214684
申请日：2014-03-15
申请人： Lyle J. Arnold
发明人： Lyle J. Arnold
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6834 , C12Q2525/161 , C12Q2525/30 , C12Q2537/162
摘要： The present invention provides methods for immobilizing target nucleic acids on a solid support utilizing combinatorial capture probe pairs. These pairs contain first and second capture oligonucleotides that each comprise a target binding region, a capture region and a stem region positioned between the target binding and capture regions. The target binding regions comprise nucleic acid sequences that allow them to hybridize to adjacent regions on the target nucleic acid. The stem regions have nucleic acid sequences that are complementary to each other and the capture regions each comprise a sequence that when positioned adjacent to one another produce a combined nucleic acid sequence that is complementary to a portion of an oligonucleotide bound to a solid support. When the first and second capture oligonucleotides are annealed to the target nucleic acid, the stem regions are brought together allowing them to hybridize, which in turn brings the capture regions together to produce a combined nucleic acid sequence. This combined nucleic acid sequence is then able to hybridize to the oligonucleotide bound to the solid support thereby immobilizing the target nucleic acid.
摘要翻译：本发明提供了使用组合捕获探针对将靶核酸固定在固体支持物上的方法。这些对包含第一和第二捕获寡核苷酸，其每个包含靶结合区，捕获区和位于靶结合区和捕获区之间的干区。靶结合区域包含允许它们与靶核酸上的相邻区域杂交的核酸序列。茎区具有彼此互补的核酸序列，并且捕获区各自包含当彼此相邻定位时产生与结合于固体支持物的寡核苷酸结合的部分互补的组合核酸序列的序列。当第一和第二捕获寡核苷酸与靶核酸退火时，将茎区域聚集在一起使得它们能够杂交，这又使得捕获区域一起产生组合的核酸序列。这种组合的核酸序列然后能够与结合到固体支持物的寡核苷酸杂交，从而固定目标核酸。

8. 发明申请

US20120264122A1 METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION 有权
标题翻译：用于核酸扩增的方法和组合物
公开(公告)号：US20120264122A1
公开(公告)日：2012-10-18
申请号：US13460341
申请日：2012-04-30
申请人： Steven T. BRENTANO , Dmitry LYAKHOV , James D. CARLSON , Norman C. NELSON , Lyle J. ARNOLD, JR.
发明人： Steven T. BRENTANO , Dmitry LYAKHOV , James D. CARLSON , Norman C. NELSON , Lyle J. ARNOLD, JR.
IPC分类号： C12P19/34 , C12Q1/68 , C07H21/04
CPC分类号： C12Q1/6881 , C12Q1/6834 , C12Q1/6844 , C12Q1/6865 , C12Q2525/155 , C12Q2525/186 , C12Q2525/197 , C12Q2531/143 , C12Q2565/543
摘要： Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.
摘要翻译：公开了用于体外核酸扩增的组合物，其包括目标特异性通用（TSU）启动子引物或启动子提供者寡核苷酸，其包括与扩增的靶序列特异性杂交的靶特异性（TS）序列和通用（U）序列，通过使用通用序列的引物引入扩增的序列。公开了体外核酸扩增方法，其中使用一种或多种TSU寡核苷酸将U序列连接到目标捕获步骤中的靶核酸，然后在基本上等温条件下进行的随后扩增步骤中使用U序列引物制备含有指示样品中靶核酸存在的U序列的扩增产物。

9. 发明授权

US07790877B2 Antisense oligonucleotides with increased RNase sensitivity 有权
标题翻译：具有增加的RNA酶敏感性的反义寡核苷酸
公开(公告)号：US07790877B2
公开(公告)日：2010-09-07
申请号：US10142566
申请日：2002-05-08
申请人： Timothy A. Riley , Bob D. Brown , Lyle J. Arnold
发明人： Timothy A. Riley , Bob D. Brown , Lyle J. Arnold
IPC分类号： C07H21/04
CPC分类号： C12N15/1093 , C12N15/113 , C12N15/1135 , C12N15/1137 , C12N2310/12 , C12N2310/31 , C12N2310/3125 , C12N2310/313 , C12N2310/315 , C12N2310/318 , C12N2310/3181 , C12N2310/321 , C12N2310/33 , C12N2310/346 , C12N2310/351 , C12N2310/3533 , C12N2310/3521
摘要： Combinatorial libraries comprise first oligonucleotide analogs and second oligonucleotide analogs which are coupled together to form antisense molecules capable of binding target polynucleotides and activating an RNase, and ribozymes capable of cleaving polynucleotides.
摘要翻译：组合文库包含第一寡核苷酸类似物和第二寡核苷酸类似物，其偶联在一起以形成能够结合靶多核苷酸并激活核糖核酸酶的反义分子，以及能够切割多核苷酸的核酶。

10. 发明授权

US06031091A Non-nucleotide linking reagents for nucleotide probes 失效
标题翻译：用于核苷酸探针的非核苷酸连接试剂
公开(公告)号：US06031091A
公开(公告)日：2000-02-29
申请号：US908535
申请日：1997-08-07
申请人： Lyle J. Arnold, Jr. , Mark A. Reynolds , Ram S. Bhatt
发明人： Lyle J. Arnold, Jr. , Mark A. Reynolds , Ram S. Bhatt
IPC分类号： C07F9/24 , C07H21/00 , C07F9/02
CPC分类号： C07H21/00 , C07F9/2408
摘要： A versatile reagent with a non-nucleotide monomeric unit having a ligand, and first and second coupling groups which are linked to the non-nucleotide monomeric unit. The ligand can be either a chemical moiety, such as a label or intercalator, or a linking arm which can be linked to such a moiety. Such reagent permits preparation of versatile nucleotide/non-nucleotide polymers, having any desired sequence of nucleotide and non-nucleotide monomeric units, each of the latter of which bear a desired ligand. These polymers can for example, be used as probes which can exhibit enhanced sensitivity and/or which are capable of detecting a genus of nucleotides each species of which has a common target nucleotide sequence of interest bridged by different sequences not of interest.
摘要翻译：具有具有配体的非核苷酸单体单元的多功能试剂和与非核苷酸单体单元连接的第一和第二偶联基团。配体可以是化学部分，例如标记或嵌入剂，或可以连接到这种部分的连接臂。这种试剂允许制备通用的核苷酸/非核苷酸聚合物，具有任何所需的核苷酸和非核苷酸单体单元序列，后者各自具有所需的配体。这些聚合物可以例如用作可以显示增强的灵敏度的探针和/或能够检测核苷酸属的核苷酸，其中每个物种具有由不感兴趣的不同序列桥接的共同目标核苷酸序列。

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