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。

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。

公开(公告)号：US06355417B2

公开(公告)日：2002-03-12

申请号：US09039061

申请日：1998-03-13

申请人： Douglas T. Gjerde ,  Paul D. Taylor

发明人： Douglas T. Gjerde ,  Paul D. Taylor

IPC分类号： C12Q168

CPC分类号： G01N27/44721 ,  G01N30/8651

摘要： A method and apparatus for representing double stranded nucleic acid fragments which have been separated by a chromatographic process as an array of bands which can be accurately quantified, optimized and stored. Using, for example, a Matched Ion Polynucleotide Chromatography (MIPC) process, an analog output from a UV detector is digitized and input to a computer. The digitized signal is converted to a linear array of bands which may be displayed on a video display terminal. The intensity and/or color of a band may correlate to the amount of double stranded nucleic acid in the respective fraction or the respective double stranded nucleic acid fragment above a user selected threshold level at a corresponding point in the digitized signal. The calculated base pair length, concentration, and retention time of each band in the array of bands may be displayed in alphanumeric form.

摘要翻译： 用于表示已经通过色谱法分离的双链核酸片段的方法和装置可以被精确量化，优化和储存的条带阵列。 使用例如匹配离子多核苷酸色谱（MIPC）方法，将来自UV检测器的模拟输出数字化并输入到计算机。 数字化信号被转换成可以显示在视频显示终端上的线性阵列阵列。 条带的强度和/或颜色可以与在数字化信号的对应点处的用户选择的阈值水平之上的相应级分或相应双链核酸片段中的双链核酸的量相关。 频带阵列中每个频带的计算的碱基对长度，浓度和保留时间可以以字母数字形式显示。

公开(公告)号：US06355165B1

公开(公告)日：2002-03-12

申请号：US09580302

申请日：2000-05-26

申请人： John E. Sutton ,  Douglas T. Gjerde ,  Paul D. Taylor

发明人： John E. Sutton ,  Douglas T. Gjerde ,  Paul D. Taylor

IPC分类号： B01D1508

CPC分类号： B01J20/287 ,  B01D15/161 ,  B01D15/20 ,  B01D15/366 ,  G01N30/30 ,  G01N30/54 ,  G01N30/96

摘要： A liquid chromatography apparatus with stationary and mobile phase temperature controls suitable for polynucleotide separations by MIPC and DMIPC processes. The apparatus includes heater means with a temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; a coil of capillary tubing having an inlet end and an outlet end. The outlet end of the capillary tubing is connected with the inlet end of the separation column. The inlet end of the capillary tubing comprising means for receiving process liquid, the tubing having a length of from 6 to 400 cm having a linear tubing length of heating means. The separation column and the coil of capillary tubing are enclosed in the heater means. The capillary tubing preferably is PEEK or titanium. The heater means can be an air batch oven. Preferably, it is a heat-conducting block having a first heat transfer surface, a separation column receptacle, and a capillary coil receptacle. A separation column is positioned within the separation column receptacle in heat conducting relationship with an inner wall thereof. A coil of capillary tubing is positioned in the capillary coil receptacle, the outer extremities of the coil being in heat conducting relationship with an inner wall of the capillary coil receptacle. Optimally, the heating means is a Peltier heating and cooling unit in heat conducting relationship with a heat transfer surface of the heating block.

摘要翻译： 具有适用于MIPC和DMIPC工艺的多核苷酸分离的固定和流动相温度控制的液相色谱仪。 该装置包括具有温度控制系统的加热器装置; 具有入口端的匹配离子多核苷酸色谱分离柱; 具有入口端和出口端的毛细管线圈。 毛细管的出口端与分离柱的入口端连接。 毛细管的入口端包括用于接收处理液体的装置，该管道具有6至400cm的长度，其具有加热装置的线性管长度。 分离柱和毛细管线圈封闭在加热器装置中。 毛细管优选为PEEK或钛。 加热器装置可以是空气分批炉。 优选地，它是具有第一传热表面，分离柱容器和毛细管线圈容器的导热块。 分离柱位于分离塔容器内，与其内壁呈导热关系。 毛细管线圈定位在毛细管线圈容器中，线圈的外端与毛细管线圈容器的内壁处于热传导关系。 最佳地，加热装置是与加热块的传热表面导热关系的珀尔帖加热和冷却单元。

公开(公告)号：US06342161B1

公开(公告)日：2002-01-29

申请号：US09481476

申请日：2000-01-11

申请人： Douglas T. Gjerde ,  Paul D. Taylor ,  Robert M. Hae Fele

发明人： Douglas T. Gjerde ,  Paul D. Taylor ,  Robert M. Hae Fele

IPC分类号： B01D1508

CPC分类号： B01D15/366

摘要： Mixtures of dsDNA fragments are separated by Matched Ion Polynucleotide Chromatography (MIPC) using an isocratic mobile phase to elute polynucleic acid from an MIPC column. The use of isocratic elution conditions provides a marked improvement in the separation of dsDNA fragments compared to gradient elution conditions. Isocratic elution can also be used to effect an improved separation of heteroduplex and homoduplex mixtures when the chromatography is performed under partially denaturing conditions. In addition, dsDNA fragments are bound to the stationary phase under isocratic conditions until a solvent concentration is reached which releases fragments of a particular base pair length range. This separation process is different from the equilibrium partitioning process observed under gradient elution conditions.

摘要翻译： 通过使用等度流动相的匹配离子多核苷酸色谱（MIPC）分离dsDNA片段的混合物以从MIPC柱洗脱多核酸。 与梯度洗脱条件相比，使用等度洗脱条件提供了dsDNA片段分离的显着改善。 当在部分变性条件下进行层析时，等度洗脱也可用于改进异源双链和同源双链混合物的分离。 此外，双链DNA片段在等度条件下与固定相结合，直至达到溶剂浓度，释放特定碱基对长度范围的片段。 该分离过程不同于在梯度洗脱条件下观察到的平衡分配过程。

公开(公告)号：US06258264B1

公开(公告)日：2001-07-10

申请号：US09399472

申请日：1999-09-20

申请人： Douglas T. Gjerde ,  Paul D. Taylor

发明人： Douglas T. Gjerde ,  Paul D. Taylor

IPC分类号： B01D1508

CPC分类号： B01J20/287 ,  B01J20/3204 ,  B01J20/3225 ,  B01J20/3227 ,  B01J20/3268 ,  B01J20/328

摘要： Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

摘要翻译： 平均直径约为0.5-100微米的无孔珠粒适用于多核苷酸混合物的色谱分离，当珠粒包含用聚合物涂覆的无孔颗粒或具有基本上全部被非极性烃封端的表面底物基团时， 取代的烃基。 珠使用匹配离子多核苷酸色谱法提供多核苷酸的有效分离。

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.

公开(公告)号：US06503397B2

公开(公告)日：2003-01-07

申请号：US09828315

申请日：2001-04-05

申请人： Douglas T. Gjerde ,  Paul D. Taylor

发明人： Douglas T. Gjerde ,  Paul D. Taylor

IPC分类号： B01D1508

CPC分类号： B01D15/366 ,  B01J20/28004 ,  B01J20/28042 ,  B01J20/287 ,  B01J20/3204 ,  B01J20/3225 ,  B01J20/3227 ,  B01J20/3268 ,  C12N15/1006 ,  C12N15/101

摘要： Nonporous beads having an average diameter of about 0.5-100 microns are suitable for chromatographic separation of mixtures of polynucleotides when the beads comprise a nonporous particle which are coated with a polymer or which have substantially all surface substrate groups endcapped with a non-polar hydrocarbon or substituted hydrocarbon group. The beads provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography.

公开(公告)号：US06461819B1

公开(公告)日：2002-10-08

申请号：US09643120

申请日：2000-08-21

申请人： Douglas T. Gjerde ,  Paul D. Taylor

发明人： Douglas T. Gjerde ,  Paul D. Taylor

IPC分类号： C12Q168

CPC分类号： C12Q1/6816 ,  C12Q2565/137 ,  C12Q2565/131

摘要： The present invention provides a method for calibrating a MIPC column wherein the calibration relates to the determination of the organic solvent component in the mobile phase required to elute dsDNA fragments of different base pair lengths at specific retention times. Since a MIPC column affords highly reproducible separations, once calibrated, the base pair length of unknown dsDNA fragments can be determined by comparing their retention times to those obtained on a standard calibration chromatogram. The standard calibration chromatogram is obtained by chromatographing a standard dsDNA ladder containing fragments of known base pair length. In addition, a method is provided to determine the presence of nicks in dsDNA using MIPC under fully denaturing conditions, e.g., 80° C. In one embodiment, this method is applied to the detection of mutations in dsDNA.

摘要翻译： 本发明提供了一种用于校准MIPC柱的方法，其中校准涉及在特定保留时间洗脱不同碱基对长度的dsDNA片段所需的流动相中有机溶剂组分的测定。 由于MIPC柱提供高度可重复的分离，一旦校准，可以通过将其保留时间与在标准校准色谱图上获得的保留时间进行比较来确定未知dsDNA片段的碱基对长度。 标准校准色谱图是通过色谱分离含有已知碱基对长度的片段的标准dsDNA梯度而获得的。 此外，提供了一种方法，用于在完全变性条件（例如80℃）下使用MIPC测定dsDNA中缺口的存在。在一个实施方案中，该方法用于检测dsDNA中的突变。

公开(公告)号：US6024878A

公开(公告)日：2000-02-15

申请号：US183047

申请日：1998-10-30

申请人： Douglas T. Gjerde ,  Paul D. Taylor ,  Robert M. Hae Fele

发明人： Douglas T. Gjerde ,  Paul D. Taylor ,  Robert M. Hae Fele

IPC分类号： C12N15/09 ,  B01D15/08 ,  B01D15/36 ,  B01J20/26 ,  B01J20/32 ,  C12N15/10 ,  C12Q1/68 ,  G01N30/34 ,  G01N30/88

CPC分类号： B01D15/366

摘要： Mixtures of dsDNA fragments are separated by Matched Ion Polynucleotide Chromatography (MIPC) using an isocratic mobile phase to elute polynucleic acid from an MIPC column. The use of isocratic elution conditions provides a marked improvement in the separation of dsDNA fragments compared to gradient elution conditions. Isocratic elution can also be used to effect an improved separation of heteroduplex and homoduplex mixtures when the chromatography is performed under partially denaturing conditions. In addition, dsDNA fragments are bound to the stationary phase under isocratic conditions until a solvent concentration is reached which releases fragments of a particular base pair length range. This separation process is different from the equilibrium partitioning process observed under gradient elution conditions.

摘要翻译： 通过使用等度流动相的匹配离子多核苷酸色谱（MIPC）分离dsDNA片段的混合物以从MIPC柱洗脱多核酸。 与梯度洗脱条件相比，使用等度洗脱条件提供了dsDNA片段分离的显着改善。 当在部分变性条件下进行层析时，等度洗脱也可用于改进异源双链和同源双链混合物的分离。 此外，双链DNA片段在等度条件下与固定相结合，直至达到溶剂浓度，释放特定碱基对长度范围的片段。 该分离过程不同于在梯度洗脱条件下观察到的平衡分配过程。