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1. 发明授权

US08399197B2 Methods for amplifying polymeric nucleic acids 有权
公开(公告)号：US08399197B2
公开(公告)日：2013-03-19
申请号：US12555967
申请日：2009-09-09
申请人： Mark Aaron Behlke , Joseph Alan Walder , Jeffrey A. Manthey
发明人： Mark Aaron Behlke , Joseph Alan Walder , Jeffrey A. Manthey
IPC分类号： C12Q1/68 , C07H21/04
CPC分类号： C12Q1/686 , C12Q1/6848 , C12Q1/6853 , C12Q2527/143 , C12Q2525/101 , C12Q2549/119 , C12Q2525/186
摘要： The invention provides compositions and methods for amplifying nucleic acid polymer sequences in a high complexity nucleic acid sample. The unique compositions of the invention include a primer set composed of a mixture of two types of primers for DNA synthesis. For extension in one direction, the primers all contain modifications that destroy their ability to serve as templates that can be copied by DNA polymerases. For extension in the opposite direction the set includes at least one primer that can serve as a template and be replicated by DNA polymerases throughout its length. The method can be carried out by mixing the nucleic acid polymer sequence of interest with the set of DNA synthesis primers in an amplification reaction mixture. The reaction mixture is then subjected to temperature cycling analogous to the temperature cycling in PCR reactions. At least one primer in the primer set hybridizes to the nucleic acid polymer. It is preferred that the non-replicable primer hybridizes to the nucleic acid polymer and is extended to produce an extension product that contains sequence from the nucleic acid polymer to which the replicable primer then hybridizes. Of course, if the nucleic acid polymer is double stranded, both the replicable and nonreplicable primers will hybridize and be extended by DNA polymerase.

2. 发明授权

US07112406B2 Polynomial amplification of nucleic acids 有权
标题翻译：核酸多项式扩增
公开(公告)号：US07112406B2
公开(公告)日：2006-09-26
申请号：US10377168
申请日：2003-02-28
申请人： Mark Aaron Behlke , Joseph Alan Walder , Jeffrey A. Manthey
发明人： Mark Aaron Behlke , Joseph Alan Walder , Jeffrey A. Manthey
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6848 , C12Q1/6853 , C12Q2549/119 , C12Q2525/186
摘要： The invention provides methods and compositions for the amplification and replication of nucleic acid molecules. In particular, novel amplification methods, referred to herein as polynomial amplification, are provided. According to these methods, a nucleic acid molecule to be amplified is contacted with at least two primers; a non-replicable primer which may hybridize to the nucleic acid molecule being amplified, and a replicable primer which may hybridize to a primer extension product generated from extension of the non-replicable primer.
摘要翻译：本发明提供用于扩增和复制核酸分子的方法和组合物。特别地，提供了新颖的扩增方法，这里称为多项式放大。根据这些方法，将要扩增的核酸分子与至少两个引物接触; 可以与被扩增的核酸分子杂交的不可复制的引物，以及可复制的引物，其可以与由不可复制的引物的延伸产生的引物延伸产物杂交。

3. 发明授权

US09434988B2 RNase H-based assays utilizing modified RNA monomers 有权
标题翻译：使用修饰的RNA单体的基于RNA酶H的测定
公开(公告)号：US09434988B2
公开(公告)日：2016-09-06
申请号：US13429077
申请日：2012-03-23
申请人： Mark Aaron Behlke , Scott D. Rose , Joseph Dobosy , Joseph Alan Walder
发明人： Mark Aaron Behlke , Scott D. Rose , Joseph Dobosy , Joseph Alan Walder
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6858 , C12Q2521/327 , C12Q2563/107
摘要： The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.
摘要翻译：本发明提供了切割核酸链以启动，辅助，监测或进行生物测定的方法。

4. 发明申请

US20120258455A1 RNase H-Based Assays Utilizing Modified RNA Monomers 有权
标题翻译：使用修饰的RNA单体的RNA酶H基测定法
公开(公告)号：US20120258455A1
公开(公告)日：2012-10-11
申请号：US13429077
申请日：2012-03-23
申请人： Mark Aaron Behlke , Scott D. Rose , Joseph Dobosy , Joseph Alan Walder
发明人： Mark Aaron Behlke , Scott D. Rose , Joseph Dobosy , Joseph Alan Walder
IPC分类号： C12P19/34 , G01N21/64 , G01N27/62 , C12Q1/68
CPC分类号： C12Q1/6858 , C12Q2521/327 , C12Q2563/107
摘要： The present invention provides methods of cleaving a nucleic acid strand to initiate, assist, monitor or perform biological assays.
摘要翻译：本发明提供了切割核酸链以启动，辅助，监测或进行生物测定的方法。

5. 发明授权

US06773885B1 Compositions and methods for visual ribonuclease detection assays 有权
标题翻译：视觉核糖核酸酶检测试验的组合物和方法
公开(公告)号：US06773885B1
公开(公告)日：2004-08-10
申请号：US09968733
申请日：2001-10-01
申请人： Joseph Alan Walder , Mark Aaron Behlke , Eric Jeffrey Devor , Lingyan Huang
发明人： Joseph Alan Walder , Mark Aaron Behlke , Eric Jeffrey Devor , Lingyan Huang
IPC分类号： C12Q168
CPC分类号： G01N33/573 , C12Q1/6844 , C12Q2565/1015 , C12Q2521/301
摘要： The present invention relates to methods for detecting the presence of ribonuclease enzymes, more specifically to methods that provide for a visual detection assay. The methods entail contacting a test sample suspected of containing ribonuclease activity with a substrate containing a ribonuclease-sensitive internucleotide linkage flanked directly or indirectly by a fluorescence reporter group and a dark quencher, such that if a ribonuclease activity is present in the sample, the ribonuclease-sensitive internucleotide linkage is cleaved and the fluorescence reporter group emits a visually detectable signal. The present invention further provides novel nucleic acid compositions used as substrates for such assays and encompasses kits for performing the methods of the invention.
摘要翻译：本发明涉及用于检测核糖核酸酶的存在的方法，更具体地涉及提供视觉检测测定法的方法。所述方法需要将怀疑含有核糖核酸酶活性的测试样品与含有核酸核酸酶敏感的核苷酸间连接物的底物接触，所述底物直接或间接地由荧光报道基团和暗猝灭剂连接，使得如果样品中存在核糖核酸酶活性，则核糖核酸酶敏感的核苷酸间键断裂，荧光报告基团发出视觉上可检测的信号。本发明还提供了用作这种测定的底物的新型核酸组合物，并且包括用于实施本发明方法的试剂盒。

6. 发明申请

US20120123751A1 Method for Estimating a Melting Temperature of a Nucleic Acid in Buffers Containing Magnesium Ions 审中-公开
标题翻译：用于估计含有镁离子的缓冲液中核酸的熔融温度的方法
公开(公告)号：US20120123751A1
公开(公告)日：2012-05-17
申请号：US13290899
申请日：2011-11-07
申请人： Richard Owczarzy , Bernardo Moreira , Yong You , Mark Aaron Behlke , Joseph Alan Walder
发明人： Richard Owczarzy , Bernardo Moreira , Yong You , Mark Aaron Behlke , Joseph Alan Walder
IPC分类号： G06F19/12 , G06F17/10
CPC分类号： G16B25/00 , G16B20/00
摘要： The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, in the presence of divalent cations, particularly duplexes of oligonucleotides which may be used as, for example, but not limited to primers or probes in PCR and/or hybridization assays. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of divalent cation salt conditions on the melting temperature.
摘要翻译：本发明涉及用于预测或估计双链核酸的解链温度的方法和系统，所述方法和系统在二价阳离子，特别是寡核苷酸的双链体存在下可用作例如但不限于PCR和/ 或杂交测定。方法和算法使用具有作为特定核苷酸序列的功能的术语和系数的新型公式来估计二价阳离子盐条件对熔融温度的影响。

7. 发明申请

US20100167353A1 RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS 有权
标题翻译：使用修饰的RNA单体的RNASE H基测定
公开(公告)号：US20100167353A1
公开(公告)日：2010-07-01
申请号：US12507142
申请日：2009-07-22
申请人： Joseph Alan Walder , Mark Aaron Behlke , Scott Rose , Joseph Dobosy
发明人： Joseph Alan Walder , Mark Aaron Behlke , Scott Rose , Joseph Dobosy
IPC分类号： C12P19/34
CPC分类号： C12Q1/6848 , C12Q1/6844 , C12Q1/6853 , C12Q1/6858 , C12Q1/686 , C12Q2549/101 , C12Q2525/186 , C12Q2525/121 , C12Q2521/301
摘要： The present invention pertains to novel oligonucleotide compounds for use in various biological assays, such as nucleic acid amplification, ligation and sequencing reactions. The novel oligonucleotides comprise a ribonucleic acid domain and a blocking group at or near the 3′ end of the oligonucleotide. These compounds offer an added level of specificity previously unseen. Methods for performing nucleic acid amplification, ligation and sequencing are also provided. Additionally, kits containing the oligonucleotides are also disclosed herein.
摘要翻译：本发明涉及用于各种生物测定的新型寡核苷酸化合物，例如核酸扩增，连接和测序反应。该寡核苷酸在寡核苷酸的3'末端或其附近包含核糖核酸结构域和封闭基团。这些化合物提供了先前未见的特异性增加水平。还提供了进行核酸扩增，连接和测序的方法。此外，本文还公开了含有寡核苷酸的试剂盒。

8. 发明申请

US20150307917A1 RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS 审中-公开
标题翻译：使用修饰的RNA单体的RNASE H基测定
公开(公告)号：US20150307917A1
公开(公告)日：2015-10-29
申请号：US14123944
申请日：2013-12-02
申请人： Joseph Alan Walder , Mark Aaron Behlke , Scott D. Rose , Joseph R. Dobosy , Susan M. Rupp
发明人： Joseph Alan Walder , Mark Aaron Behlke , Scott D. Rose , Joseph R. Dobosy , Susan M. Rupp
IPC分类号： C12Q1/68 , C12N9/22
CPC分类号： C12Q1/686 , C12N9/22 , C12Q1/6844 , C12Y301/26004 , C12Q2521/327 , C12Q2525/186
摘要： Methods and compositions are provided for improving specificity during amplification of a target DNA sequence. The methods and compositions rely upon the use of an RNase H enzyme, a polymerase, and RNase H enzyme-sensitive, blocked-cleavable oligonucleotide primers in the amplification reactions, wherein the reaction mixtures include either an optimized final concentration of a divalent metal salt comprising 2.0 mM or less of free Mg++ cation and/or an optimized final concentration of a non-ionic detergent comprising at least about 0.001% polyethylene glycol hexadecyl ether.
摘要翻译：提供了用于在扩增靶DNA序列期间改善特异性的方法和组合物。所述方法和组合物依赖于在扩增反应中使用RNase H酶，聚合酶和RNase H酶敏感的可封闭切割的寡核苷酸引物，其中反应混合物包括优化的最终浓度的二价金属盐，其包含 2.0mM或更少的游离Mg ++阳离子和/或优选的最终浓度的非离子洗涤剂，其包含至少约0.001％的聚乙二醇十六烷基醚。

9. 发明申请

US20090198453A1 METHOD FOR ESTIMATING A MELTING TEMPERATURE OF A NUCLEIC ACID IN BUFFERS CONTAINING MAGNESIUM IONS 有权
标题翻译：用于估计含有镁离子的缓冲液中核酸的熔融温度的方法
公开(公告)号：US20090198453A1
公开(公告)日：2009-08-06
申请号：US11970509
申请日：2008-01-07
申请人： Richard Owczarzy , Bernardo Moreira , Yong You , Mark Aaron Behlke , Joseph Alan Walder
发明人： Richard Owczarzy , Bernardo Moreira , Yong You , Mark Aaron Behlke , Joseph Alan Walder
IPC分类号： G06F19/00
CPC分类号： G06F19/20 , G06F19/18
摘要： The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, in the presence of divalent cations, particularly duplexes of oligonucleotides which may be used as, for example, but not limited to primers or probes in PCR and/or hybridization assays. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of divalent cation salt conditions on the melting temperature.
摘要翻译：本发明涉及用于预测或估计双链核酸的解链温度的方法和系统，所述方法和系统在二价阳离子，特别是寡核苷酸的双链体存在下可用作例如但不限于PCR和/ 或杂交测定。方法和算法使用具有作为特定核苷酸序列的功能的术语和系数的新型公式来估计二价阳离子盐条件对熔融温度的影响。

10. 发明授权

US06889143B2 Methods and systems for estimating the melting temperature (TM) for polynucleotide molecules 有权
标题翻译：用于估计多核苷酸分子的熔解温度（TM）的方法和系统
公开(公告)号：US06889143B2
公开(公告)日：2005-05-03
申请号：US10660253
申请日：2003-09-11
申请人： Mark Aaron Behlke , Lingyan Huang , Richard Owczarzy , Joseph Alan Walder
发明人： Mark Aaron Behlke , Lingyan Huang , Richard Owczarzy , Joseph Alan Walder
IPC分类号： C12Q1/68 , G01N20060101 , G01N33/48 , G01N33/50 , G06F17/00 , G06F17/10 , G06F17/11 , G06F19/20 , G06F19/22
CPC分类号： G06F19/20 , C12Q1/6813 , G06F19/22
摘要： The invention relates to methods and systems for predicting or estimating the melting temperature of duplex nucleic acids, particularly duplexes of oligonucleotides which may be used, for example, as primers or probes in PCR and/or hybridization assays. The invention also relates to methods and systems for designing and selecting oligonucleotide probes and primers having a predicted melting temperature which is optimized for such assays. To this end, algorithms and methods are provided for predicting the melting temperature of a nucleic acid having a predetermined sequence. These methods and algorithms estimate the melting temperature of a nucleic acid duplex under particular salt conditions. The methods and algorithms use novel formulas, having terms and coefficients that are functions of the particular nucleotide sequence, to estimate the effect of particular salt conditions on the melting temperature. As such, the methods and systems of the invention provide superior result compared to existing methods, which do not consider sequence dependent effects of changing salt conditions.
摘要翻译：本发明涉及用于预测或估计双链体核酸，特别是寡核苷酸双链体的解链温度的方法和系统，其可用于例如PCR和/或杂交测定中的引物或探针。本发明还涉及用于设计和选择寡核苷酸探针和引物的方法和系统，所述寡核苷酸探针和引物具有针对这种测定法优化的预测的熔融温度。为此，提供了用于预测具有预定序列的核酸的解链温度的算法和方法。这些方法和算法估计在特定盐条件下核酸双链体的解链温度。方法和算法使用具有作为特定核苷酸序列的功能的术语和系数的新型公式来估计特定盐条件对熔融温度的影响。因此，与现有方法相比，本发明的方法和系统提供了优异的结果，其不考虑改变盐条件的序列依赖性影响。

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