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1. 发明授权

US06576133B2 Method and system for RNA analysis by matched ion polynucleotide chromatography 失效
标题翻译：通过匹配离子多核苷酸色谱法进行RNA分析的方法和系统
公开(公告)号：US06576133B2
公开(公告)日：2003-06-10
申请号：US09753856
申请日：2001-01-02
申请人： Douglas T. Gjerde , David P. Hornby , Christopher P. Hanna , Alexander I. Kuklin , Robert M. Haefele , Paul D. Taylor
发明人： Douglas T. Gjerde , David P. Hornby , Christopher P. Hanna , Alexander I. Kuklin , Robert M. Haefele , Paul D. Taylor
IPC分类号： B01D1508
CPC分类号： B01D15/366 , C12N15/101
摘要： A Matched Ion Polynucleotide Chromatography method and system for size-based segregation of a mixture of RNA molecules. The method includes applying the mixture to a polymeric separation medium having non-polar surfaces and eluting the RNA molecules with a mobile phase which includes counterion reagent and an organic component. The preferred surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation. The elution is preferably performed at a temperature sufficient to denature the RNA. The method can be used in segregating RNA molecules having lengths in the range of about 100 to 20,000 nucleotides. Improved segregation is obtained using a chromatography column having an ID greater than about 5 mm. Examples of separation media include beads and monolithic columns.
摘要翻译：匹配的离子多核苷酸色谱法和基于尺寸的RNA分子混合物分离的系统。该方法包括将混合物施加到具有非极性表面的聚合物分离介质中，并用包含抗衡离子试剂和有机组分的流动相洗脱RNA分子。优选的表面的特征在于基本上不含可以干扰RNA分离的多价阳离子。洗脱优选在足以使RNA变性的温度下进行。该方法可用于分离长度在约100至20,000个核苷酸范围内的RNA分子。使用ID大于约5mm的色谱柱获得改进的分离。分离介质的实例包括珠粒和整体柱。

2. 发明授权

US06521123B2 Polynucleotide separations on polymeric separation media 失效
标题翻译：聚合物分离介质上的多核苷酸分离
公开(公告)号：US06521123B2
公开(公告)日：2003-02-18
申请号：US09848385
申请日：2001-05-02
申请人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
发明人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
IPC分类号： B01D1508
CPC分类号： B01D15/366 , C12N15/101
摘要： Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surfaces of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from mutivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched Ion Polynucleolide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.
摘要翻译：非极性聚合物分离介质，例如珠粒或整料适用于当介质的表面未被取代或被具有1至1,000,000个碳原子的烃基取代时的多核苷酸混合物的色谱分离，并且当表面基本上不含多价阳离子污染聚合物介质使用匹配离子多核苷酸色谱法提供多核苷酸的有效分离。维持和储存聚合物介质的方法包括用多价阳离子结合剂进行处理。

3. 发明授权

US06838242B2 Modifying double stranded DNA to enhance separations by matched ion polynucleotide chromatography 失效
标题翻译：通过匹配的离子多核苷酸色谱法修饰双链DNA以增强分离
公开(公告)号：US06838242B2
公开(公告)日：2005-01-04
申请号：US09826055
申请日：2001-04-03
申请人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
发明人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
IPC分类号： B01D15/00 , C07H21/02 , C07H21/04 , C12M1/34 , C12P19/34 , C12Q1/68 , G01N33/00
CPC分类号： C12Q1/6816 , C12Q1/6827 , Y10T436/143333 , C12Q2565/137 , C12Q2527/107
摘要： Covalently bound non-polar tags are used to increase the retention times of double stranded polynucleotides on Matched Ion Polynucleotide Chromatography (MIPC) columns. In doing so, separations of DNA mixture components is improved. Additionally, when the non-polar tags are fluorophores, detection limits are also greatly reduced. Strategically tagged primers are used in conduction with PCR to produce DNA fragments having specifically tagged strands. This improves mutation detection by MIPC in several ways. Separations are improved, detection sensitivity is enhanced, and non-stoichiometric addition of wild type DNA prior to hybridization is now possible since only tagged fragments will be observed with a fluorescence detector. Non-polar tags are also used as a novel alternative to G-C clamping during MIPC under partially denaturing conditions. Reversible DNA binding dyes, such as DNA intercalator dyes and DNA groove binding dyes, are used to reduce the detection limit of polynucleotides separated by MIPC.

4. 发明授权

US5986085A Matched ion polynucleotide chromatography (MIPC) process for separation of polynucleotide fragments 失效
标题翻译：用于分离多核苷酸片段的匹配离子多核苷酸色谱（MIPC）方法
公开(公告)号：US5986085A
公开(公告)日：1999-11-16
申请号：US65913
申请日：1998-04-24
申请人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
发明人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
IPC分类号： C12N15/10 , C07H21/00 , C07H21/02 , C07H21/04
CPC分类号： C12N15/101
摘要： A batch process for obtaining polynucleotide fragments, such as dsDNA, having a selected size from a mixture of polynucleotide fragments including the steps of a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface; b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium. The binding medium can be organic polymer or inorganic particle beads. The mixture of polynucleotides can be the product of a PCR amplification. The binding medium can be contained within a column, a web or a container.
摘要翻译：用于从多核苷酸片段的混合物中获得具有选定大小的多核苷酸片段（例如dsDNA）的分批方法，包括以下步骤：a）将多核苷酸片段和抗衡离子混合物的溶液施用于具有疏水性表面的结合介质; b）使所述结合介质与第一剥离溶剂和抗衡离子剂接触，所述第一剥离溶剂具有足以从所述结合介质释放的有机组分的浓度，所述多个片段的尺寸小于所选择的尺寸，并且除去所述第一剥离溶剂从结合介质; 和c）使所述结合介质与第二剥离溶剂接触，所述第二剥离溶剂具有足以从所述结合介质释放具有所选择尺寸的多核苷酸片段的有机组分浓度，以及从所述结合介质中除去所述第二剥离溶剂。结合介质可以是有机聚合物或无机颗粒珠。多核苷酸的混合物可以是PCR扩增的产物。结合介质可以包含在柱，网或容器中。

5. 发明授权

US07138518B1 Liquid chromatographic separation of polynucleotides 有权
标题翻译：多核苷酸的液相色谱分离
公开(公告)号：US07138518B1
公开(公告)日：2006-11-21
申请号：US09714579
申请日：2000-11-16
申请人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor , Christopher P. Hanna , Alezander I. Kuklin , David P. Hornby
发明人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor , Christopher P. Hanna , Alezander I. Kuklin , David P. Hornby
IPC分类号： C07H21/00 , C12Q1/68 , B01D15/00 , B01D15/08
CPC分类号： C12N15/101
摘要： In one aspect, the invention provides a method for separating a mixture of polynucleotides, such as DNA or RNA, including (a) applying the mixture to a polymeric separation medium having non-polar surfaces, wherein the surfaces are characterized by being substantially free from multivalent cations, such as metal ions, which are free to interfere with polynucleotide separation, and (b) eluting the mixture with a mobile phase containing organic solvent and counter ion agent. In the separation of single-stranded polynucleotides, improved separation is obtained at a temperature effective to fully denature secondary structure within the polynucleotides.
摘要翻译：一方面，本发明提供了分离多核苷酸（例如DNA或RNA）的混合物的方法，包括（a）将该混合物施用于具有非极性表面的聚合物分离介质，其中所述表面的特征在于基本上不含可自由干扰多核苷酸分离的多价阳离子，例如金属离子，和（b）用含有机溶剂和抗衡离子剂的流动相洗脱该混合物。在单链多核苷酸的分离中，在有效使多核苷酸内完全变性二级结构的温度下获得改进的分离。

6. 发明授权

US06642374B2 Process for separation of polynucleotide fragments 失效
标题翻译：分离多核苷酸片段的方法
公开(公告)号：US06642374B2
公开(公告)日：2003-11-04
申请号：US09764041
申请日：2001-01-16
申请人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
发明人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
IPC分类号： C07H2100
CPC分类号： C12Q1/6806 , B01D15/366 , B01J20/26 , B01J20/264 , B01J20/28004 , B01J20/286 , B01J20/3092 , B01J20/3204 , B01J20/321 , B01J20/3227 , B01J20/3246 , B01J20/3268 , B01J2220/54 , B01J2220/58 , C12N15/101
摘要： A batch process for obtaining polynucleotide fragments, such as dsDNA, having a selected size from a mixture of polynucleotide fragments including the steps of a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface; b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium. The binding medium can be organic polymer or inorganic particle beads. The mixture of polynucleotides can be the product of a PCR amplification. The binding medium can be contained within a column, a web or a container.
摘要翻译：用于从多核苷酸片段的混合物中获得具有选定大小的多核苷酸片段（例如dsDNA）的分批方法，包括以下步骤：a）将多核苷酸片段和抗衡离子混合物的溶液施用于具有疏水性表面的结合介质; b）使所述结合介质与第一剥离溶剂和抗衡离子剂接触，所述第一剥离溶剂具有足以从所述结合介质释放的有机组分的浓度，所述多个片段的尺寸小于所选择的尺寸，并且除去所述第一剥离溶剂从结合介质; 和c）使所述结合介质与第二剥离溶剂接触，所述第二剥离溶剂具有足以从所述结合介质释放具有所选择尺寸的多核苷酸片段的有机组分浓度，以及从所述结合介质中除去所述第二剥离溶剂。结合介质可以是有机聚合物或无机颗粒珠。多核苷酸的混合物可以是PCR扩增的产物。结合介质可以包含在柱，网或容器中。

7. 发明授权

US06287822B1 Mutation detection method 有权
标题翻译：突变检测方法
公开(公告)号：US06287822B1
公开(公告)日：2001-09-11
申请号：US09129105
申请日：1998-08-04
申请人： Douglas T Gjerde , Paul D. Taylor , Robert M. Haefele
发明人： Douglas T Gjerde , Paul D. Taylor , Robert M. Haefele
IPC分类号： C12P1934
CPC分类号： C12Q1/6806 , C12Q1/6827 , C12Q2565/137 , C12Q2527/107
摘要： The present invention is directed to improved methods for detection of mutations in DNA using Denaturing Matched Ion Polynucleotide Chromatography (DMIPC). The invention includes the following aspects: analysis of PCR amplification products to identify factors that affect PCR replication fidelity; design of PCR primers; selection of an optimal temperature for performing DMIPC; selection of the mobile phase composition for gradient elution; methods for column preparation and maintenance; and methods for preparing polynucleotide samples prior to chromatographic analysis.
摘要翻译：本发明涉及使用变性匹配离子多核苷酸色谱（DMIPC）检测DNA突变的改进方法。本发明包括以下几个方面：分析PCR扩增产物，鉴定影响PCR复制保真度的因素; PCR引物设计; 选择用于执行DMIPC的最佳温度; 选择流动相组成进行梯度洗脱; 柱准备和维护方法; 以及在色谱分析之前制备多核苷酸样品的方法。

8. 发明授权

US06177559B1 Process for separation of polynucleotide fragments 有权
标题翻译：分离多核苷酸片段的方法
公开(公告)号：US06177559B1
公开(公告)日：2001-01-23
申请号：US09391963
申请日：1999-09-08
申请人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
发明人： Douglas T. Gjerde , Robert M. Haefele , Paul D. Taylor
IPC分类号： C07H2100
CPC分类号： C12Q1/6806 , B01D15/366 , B01J20/26 , B01J20/264 , B01J20/28004 , B01J20/286 , B01J20/3092 , B01J20/3204 , B01J20/321 , B01J20/3227 , B01J20/3246 , B01J20/3268 , B01J2220/54 , B01J2220/58 , C12N15/101
摘要： A batch process for obtaining polynucleotide fragments, such as dsDNA, having a selected size from a mixture of polynucleotide fragments including the steps of a) applying a solution of the mixture of polynucleotide fragments and a counterion agent to a binding medium having a hydrophobic surface; b) contacting the binding medium with a first stripping solvent and counterion agent, the first stripping solvent having a concentration of organic component sufficient to release from the binding medium all polynucleotide fragments having a size smaller than the selected size, and removing the first stripping solvent from the binding medium; and c) contacting the binding medium with a second stripping solvent having a concentration of organic component sufficient to release from the binding medium the polynucleotide fragments having the selected size, and removing the second stripping solvent from the binding medium. The binding medium can be organic polymer or inorganic particle beads. The mixture of polynucleotides can be the product of a PCR amplification. The binding medium can be contained within a column, a web or a container.
摘要翻译：用于从多核苷酸片段的混合物中获得具有选定大小的多核苷酸片段（例如dsDNA）的分批方法，包括以下步骤：a）将多核苷酸片段和抗衡离子混合物的溶液施用于具有疏水性表面的结合介质; b）使所述结合介质与第一剥离溶剂和抗衡离子剂接触，所述第一剥离溶剂具有足以从所述结合介质释放的有机组分的浓度，所述多个片段的尺寸小于所选择的尺寸，并且除去所述第一剥离溶剂从结合介质; 和c）使所述结合介质与第二剥离溶剂接触，所述第二剥离溶剂具有足以从所述结合介质释放具有所选择尺寸的多核苷酸片段的有机组分浓度，以及从所述结合介质中除去所述第二剥离溶剂。结合介质可以是有机聚合物或无机颗粒珠。多核苷酸的混合物可以是PCR扩增的产物。结合介质可以包含在柱，网或容器中。

9. 发明授权

US06136195A MIPC column cleaning system and process 失效
公开(公告)号：US06136195A
公开(公告)日：2000-10-24
申请号：US285331
申请日：1999-04-02
申请人： Paul D. Taylor , Robert M. Haefele , Douglas T. Gjerde , Kimberly A. Lamb
发明人： Paul D. Taylor , Robert M. Haefele , Douglas T. Gjerde , Kimberly A. Lamb
IPC分类号： B01D15/08
CPC分类号： B01D15/366 , B01D15/203 , G01N30/50 , G01N2030/201 , G01N2030/202
摘要： An apparatus for effecting base pair length separations of DNA fragments by matched ion paired chromatography comprising a separation column containing separation media having non-polar DNA separation surfaces, separation solution supply means, and a separation solution conduit communicating with the separation column and the separation solution supply means, and a cleaning solution valve means positioned in the separation solution conduit for injecting cleaning solution into the separation solution conduit. A process for cleaning the non-polar DNA separation surfaces in the apparatus comprising interrupting the flow of separation solvent with a block of cleaning solution injected into the flow of separation solution passing to the column, the cleaning solution containing agent which removes accumulated residues from the non-polar surface. The cleaning solution can have an alkaline pH and contain a chelating agent such as EDTA.

10. 发明授权

US06355791B1 Polynucleotide separations on polymeric separation media 有权
标题翻译：聚合物分离介质上的多核苷酸分离
公开(公告)号：US06355791B1
公开(公告)日：2002-03-12
申请号：US09562069
申请日：2000-05-01
申请人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
发明人： Douglas T. Gjerde , Paul D. Taylor , Robert M. Haefele
IPC分类号： C07H2104
CPC分类号： B01D15/366 , B01D15/20 , B01J20/285 , C08F8/02 , C08F8/30 , C08F212/08 , C08F230/08 , C12N15/101
摘要： Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surface of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from multivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.
摘要翻译：非极性聚合物分离介质，例如珠粒或整料，适用于当介质的表面未被取代或被具有1至1,000,000个碳原子的烃基取代时，多个核苷酸混合物的色谱分离，并且当表面基本上不含多价阳离子污染聚合物介质使用匹配离子多核苷酸色谱法提供多核苷酸的有效分离。维持和储存聚合物介质的方法包括用多价阳离子结合剂进行处理。

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