Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
    1.
    发明授权
    Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule 有权
    用于鉴定核酸分子中一个或多个变体核苷酸序列的方法

    公开(公告)号:US07749708B2

    公开(公告)日:2010-07-06

    申请号:US11854181

    申请日:2007-09-12

    IPC分类号: C12Q1/68 C07H21/02 C07H21/04

    摘要: The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3′ side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3′-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

    摘要翻译: 本发明涉及鉴定核酸分子中一个或多个变体核苷酸序列的方法。 该方法包括切割含有错配特异性内切核酸酶的错配的双链核酸分子,其在错配的3'侧切割,并通过将3' 跨过所述变体核苷酸。 因为变体核苷酸与接头直接相邻,所以可以容易地进行PCR和/或逐个序列分析。

    Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
    2.
    发明申请
    Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule 有权
    用于鉴定核酸分子中一个或多个变体核苷酸序列的方法

    公开(公告)号:US20090068652A1

    公开(公告)日:2009-03-12

    申请号:US11854181

    申请日:2007-09-12

    IPC分类号: C12Q1/68

    摘要: The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3′ side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a double-stranded linker with a 3′-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.

    摘要翻译: 本发明涉及鉴定核酸分子中一个或多个变体核苷酸序列的方法。 该方法包括切割含有错配特异性内切核酸酶的错配的双链核酸分子,其在错配的3'侧切割,并通过将3' 跨过所述变体核苷酸。 因为变体核苷酸与接头直接相邻,所以可以容易地进行PCR和/或逐个序列分析。

    MIPC column cleaning system and process
    5.
    发明授权
    MIPC column cleaning system and process 失效
    MIPC柱清洗系统及工艺

    公开(公告)号:US06485648B1

    公开(公告)日:2002-11-26

    申请号:US09696610

    申请日:2000-10-25

    IPC分类号: B01D1508

    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.

    摘要翻译: 一种用于通过配对离子配对色谱法实现DNA片段的碱基对长度分离的装置,其包括含有具有非极性DNA分离表面的分离介质的分离柱,分离溶液供应装置和与分离柱和分离溶液连通的分离溶液管道 供应装置和位于分离溶液管道中的清洁溶液阀装置,用于将清洁溶液注入到分离溶液导管中。 一种用于清洁设备中的非极性DNA分离表面的方法,包括用注入到通过塔的分离溶液流中的清洁溶液块中断分离溶剂的流动,该清洗溶液包含试剂从 非极性表面。 清洁溶液可以具有碱性pH并含有螯合剂如EDTA。

    Polynucleotide separations on polymeric separation media
    6.
    发明授权
    Polynucleotide separations on polymeric separation media 有权
    聚合物分离介质上的多核苷酸分离

    公开(公告)号:US06482317B2

    公开(公告)日:2002-11-19

    申请号:US09828427

    申请日:2001-04-05

    IPC分类号: B01D1508

    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 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。

    Method of concentrating polynucleotides using MIPC
    7.
    发明授权
    Method of concentrating polynucleotides using MIPC 失效
    使用MIPC浓缩多核苷酸的方法

    公开(公告)号:US06455692B1

    公开(公告)日:2002-09-24

    申请号:US09698938

    申请日:2000-10-26

    IPC分类号: C07H2104

    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。

    Apparatus and method for separating and purifying polynucleotides
    9.
    发明授权
    Apparatus and method for separating and purifying polynucleotides 有权
    用于分离和纯化多核苷酸的装置和方法

    公开(公告)号:US06265168B1

    公开(公告)日:2001-07-24

    申请号:US09318407

    申请日:1999-05-25

    IPC分类号: C12Q168

    CPC分类号: B01D15/366 B01J20/287

    摘要: A method for removing a target DNA fragment having a predetermined base-pair length from a mixture of DNA fragments comprises the following steps. A mixture of DNA fragments which may contain the target DNA fragments is applied to a separation column containing media having a nonpolar, nonporous surface, the mixture of DNA fragments being in a first solvent mixture containing a counterion and a DNA binding concentration of driving solvent in a cosolvent. The target DNA fragments are separated from the media by contacting it with a second solvent solution containing a counterion and a concentration of driving solvent in cosolvent which has been predetermined to remove DNA fragments having the target DNA fragment base pair length from the media. The target DNA fragments can be collected and optionally amplified. When the method is being applied to collect a putative fragment, if present, no DNA fragments having the base pair length of the target DNA could be present in the mixture. Alternatively, DNA fragments having the base pair length of the target DNA are present in the mixture. The disclosure also describes an ambient or low pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises a tube having an upper solution input chamber, a lower eluant receiving chamber, and a fixed unit of separation media supported therein. The separation media has nonpolar separation surfaces which are free from multivalent cations which would react with counterion to form an insoluble polar coating on the surface of the separation media.

    摘要翻译: 从DNA片段的混合物中除去具有预定碱基对长度的靶DNA片段的方法包括以下步骤。 将含有靶DNA片段的DNA片段的混合物应用于含有非极性,无孔表面的培养基的分离塔,DNA片段的混合物在含有抗衡离子的第一溶剂混合物和驱动溶剂的DNA结合浓度 共溶剂 通过使目标DNA片段与含有抗衡离子的溶剂溶液和在助溶剂中的驱动溶剂浓度的第二溶剂溶液接触来分离,所述助溶剂已被预先从介质中除去具有靶DNA片段碱基对长度的DNA片段。 可以收集目标DNA片段并任选地扩增。 当该方法被应用于收集假定片段时(如果存在),则在混合物中不能存在具有目标DNA碱基对长度的DNA片段。 或者,具有靶DNA的碱基对长度的DNA片段存在于混合物中。 本公开还描述了用于从多核苷酸片段的混合物分离多核苷酸片段的环境或低压装置,包括具有上溶液输入室,下洗脱液接收室和支撑在其中的分离介质的固定单元的管。 分离介质具有非极性分离表面,其不含多价阳离子,其将与抗衡离子反应以在分离介质的表面上形成不溶性极性涂层。

    Modifying double stranded DNA to enhance separations by matched ion polynucleotide chromatography
    10.
    发明授权
    Modifying double stranded DNA to enhance separations by matched ion polynucleotide chromatography 失效
    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:US06210885B1

    公开(公告)日:2001-04-03

    申请号:US09169440

    申请日:1998-10-09

    IPC分类号: C12Q168

    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.

    摘要翻译: 共价结合的非极性标签用于增加匹配离子多核苷酸色谱(MIPC)色谱柱上双链多核苷酸的保留时间。 在这样做时,提高了DNA混合物组分的分离。 此外,当非极性标签是荧光团时,检测限也大大降低。 策略性标记的引物用于PCR导入以产生具有特异性标记的链的DNA片段。 这可以通过多种方式改善MIPC的突变检测。 分离改善,检测灵敏度提高,杂交前非化学计量添加野生型DNA是可能的,因为只有标记的片段才能用荧光检测器观察到。 非极性标签也被用作MIPC在部分变性条件下G-C夹持的新型替代物。 使用可逆DNA结合染料,例如DNA嵌入剂染料和DNA沟槽结合染料,以降低由MIPC分离的多核苷酸的检测限。