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    Nucleic acid detection with separation
    1.
    发明授权
    Nucleic acid detection with separation 失效
    分离核酸检测

    公开(公告)号：US6054266A

    公开(公告)日：2000-04-25

    申请号：US276139

    申请日：1988-11-23

    申请人： Mel N. Kronick ,  Douglas H. Keith ,  Lincoln J. McBride ,  Norman M. Whiteley ,  Michael W. Hunkapiller

    发明人： Mel N. Kronick ,  Douglas H. Keith ,  Lincoln J. McBride ,  Norman M. Whiteley ,  Michael W. Hunkapiller

    IPC分类号： G01N33/50 ,  C12Q1/68 ,  G01N30/88 ,  G01N33/58 ,  C07H21/04

    CPC分类号： C12Q1/6813 ,  C12Q1/6804 ,  C12Q1/6823

    摘要： Nucleic acid sequences are detected by a multi-step process, involving labeling sample nucleic acid sequences, duplexing the labeled sample with a probe having a coupling element, immobilizing all of the duplexed probe and target sequence and unduplexed probe, separating specifically immobilized nucleic acid from free and non-specifically immobilized nucleic acid, releasing specifically immobilized nucleic acid, and detecting the presence of the sequence of interest by means of the label. The labeled sequence may be characterized by sizing, e.g. electrophoresis. The method provides for a sensitive and rapid means for accurate detection of sequences of interest in a wide variety of situations.

    摘要翻译： 通过多步法检测核酸序列，包括标记样品核酸序列，用具有偶联元件的探针双链化标记的样品，固定所有双链体探针和靶序列和未折射的探针，将特异性固定的核酸与 游离和非特异性固定化的核酸，释放特异性固定化的核酸，并通过标记物检测感兴趣的序列的存在。 标记序列的特征可以是尺寸，例如 电泳。 该方法提供用于在各种情况下精确检测感兴趣序列的敏感且快速的方法。

    Detection of specific sequences in nucleic acids
    4.
    发明授权
    Detection of specific sequences in nucleic acids 失效
    检测核酸中的特定序列

    公开(公告)号：US5521065A

    公开(公告)日：1996-05-28

    申请号：US257219

    申请日：1994-06-08

    申请人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    发明人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    IPC分类号： C12Q1/68 ,  C07H21/04 ,  C12P19/34

    CPC分类号： C12Q1/6862 ,  C12Q1/6813 ,  C12Q1/6827 ,  Y10S435/803 ,  Y10S435/81

    摘要： A method of testing for the presence or absence of a target sequence in a mixture of single-stranded nucleic acid fragments is disclosed. The method involves reacting a mixture of single-stranded nucleic acid fragments with a first probe which is complementary to a first region of the target sequence, and with a second probe which is complementary to a second region of the target sequence, where the first and second target regions are contiguous with one another, under hybridization conditions in which the two probes become stably hybridized to their associated target regions. Following hybridization, any of the first and second probes hybridized to contiguous first and second target regions are ligated, and the sample is tested for the presence of expected probe ligation product. The presence of ligated product indicates that the target sequence is present in the sample.

    摘要翻译： 公开了在单链核酸片段的混合物中测试靶序列的存在或不存在的方法。 该方法包括使单链核酸片段的混合物与与靶序列的第一区域互补的第一探针和与靶序列的第二区域互补的第二探针反应，其中第一和 在杂交条件下，第二目标区域彼此连续，其中两个探针稳定地与其相关联的靶区域杂交。 杂交后，将与连续的第一和第二靶区域杂交的任何第一和第二探针连接，并测试样品中是否存在预期的探针连接产物。 连接产物的存在表明靶序列存在于样品中。

    Detection of specific sequences in nucleic acids
    5.
    发明授权
    Detection of specific sequences in nucleic acids 失效
    检测核酸中的特定序列

    公开(公告)号：US5962223A

    公开(公告)日：1999-10-05

    申请号：US574826

    申请日：1995-12-19

    申请人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    发明人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    IPC分类号： C12Q1/68 ,  C07H21/04 ,  C12P19/34

    CPC分类号： C12Q1/6862 ,  C12Q1/6813 ,  C12Q1/6827 ,  Y10S435/803 ,  Y10S435/81

    摘要： The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.

    摘要翻译： 本发明提供了一种用于诊断遗传异常或其他遗传病症的方法，其可以容易地自动化。 该方法用于确定变性核酸样品中靶序列的存在或不存在，并且需要将样品与与靶序列（诊断探针）的诊断部分互补的探针杂交，并且与探针互补 在诊断探针保持基本上仅与包含靶序列的样品核酸结合的条件下，与诊断部分（连续探针）连续的核苷酸序列。 然后将诊断探针和连续探针共价连接以产生与靶序列互补的靶探针，并且除去未连接的探针。 在优选模式中，其中一个探针被标记，使得靶序列的存在或不存在可以通过熔化样品核酸 - 靶标探针双链体，洗脱解离的靶标探针和测试标签来测试。 在另一个实施方案中，通过将钩连接到未标记的探针上，而无需首先除去未共价连接的探针，从而可以通过捕捉钩来回收标记的靶探针而进行测试。 在这两种情况下，诊断探针和连续探针的存在是标签出现在测定中所必需的。 然后将上述方法应用于遗传疾病的检测。

    Detection of specific sequences in nucleic acids
    6.
    发明授权
    Detection of specific sequences in nucleic acids 失效
    检测核酸中的特异性序列

    公开(公告)号：US5242794A

    公开(公告)日：1993-09-07

    申请号：US361407

    申请日：1989-06-05

    申请人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    发明人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6862 ,  C12Q1/6813 ,  C12Q1/6827 ,  Y10S435/803 ,  Y10S435/81

    摘要： The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.

    摘要翻译： 本发明提供了一种用于诊断遗传异常或其他遗传病症的方法，其可以容易地自动化。 该方法用于确定变性核酸样品中靶序列的存在或不存在，并且需要将样品与与靶序列（诊断探针）的诊断部分互补的探针杂交，并且与探针互补 在诊断探针保持基本上仅与包含靶序列的样品核酸结合的条件下，与诊断部分（连续探针）连续的核苷酸序列。 然后将诊断探针和连续探针共价连接以产生与靶序列互补的靶探针，并且除去未连接的探针。 在优选模式中，其中一个探针被标记，使得靶序列的存在或不存在可以通过熔化样品核酸 - 靶标探针双链体，洗脱解离的靶标探针和测试标签来测试。 在另一个实施方案中，通过将钩连接到未标记的探针上，而无需首先除去未共价连接的探针，从而可以通过捕捉钩来回收标记的靶探针而进行测试。 在这两种情况下，诊断探针和连续探针的存在是标记出现在测定中所必需的。 然后将上述方法应用于遗传疾病的检测。

    Detection of specific sequences in nucleic acids
    7.
    发明授权
    Detection of specific sequences in nucleic acids 失效
    检测核酸中的特定序列

    公开(公告)号：US4883750A

    公开(公告)日：1989-11-28

    申请号：US681055

    申请日：1984-12-13

    申请人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    发明人： Norman M. Whiteley ,  Michael W. Hunkapiller ,  Alexander N. Glazer

    IPC分类号： G01N33/50 ,  C07H21/04 ,  C12Q1/00 ,  C12Q1/68

    CPC分类号： C12Q1/6862 ,  C12Q1/6816 ,  C12Q1/6827 ,  C12Q2600/156 ,  Y10S435/803 ,  Y10S436/811

    摘要： The invention provides a method for diagnosis of genetic abnormalities or other genetic conditions which can be readily automated. The method is used to determine the presence or absence of a target sequence in a sample of denatured nucleic acid and entails hybridizing the sample with a probe complementary to a diagnostic portion of the target sequence (the diagnostic probe), and with a probe complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the sample nucleic acid containing the target sequence. The diagnostic probe and contiguous probe are then covalently attached to yield a target probe which is complementary to the target sequence, and the probes which are not attached are removed. In the preferred mode, one of the probes is labeled so that the presence or absence of the target sequence can then be tested by melting the sample nucleic acid-target probe duplex, eluting the dissociated target probe, and testing for the label. In another embodiment, the testing is accomplished without first removing probes not covalently attached, by attaching a hook to the probe that is not labeled, so that the labeled target probe may be recovered by catching the hook. In both instances, the presence of both the diagnostic probe and the contiguous probe is required for the label to appear in the assay. The above method is then applied to the detection of genetic diseases.

    摘要翻译： 本发明提供了一种用于诊断遗传异常或其他遗传病症的方法，其可以容易地自动化。 该方法用于确定变性核酸样品中靶序列的存在或不存在，并且需要将样品与与靶序列（诊断探针）的诊断部分互补的探针杂交，并且与探针互补 在诊断探针保持基本上仅与包含靶序列的样品核酸结合的条件下，与诊断部分（连续探针）连续的核苷酸序列。 然后将诊断探针和连续探针共价连接以产生与靶序列互补的靶探针，并且除去未连接的探针。 在优选模式中，其中一个探针被标记，使得靶序列的存在或不存在可以通过熔化样品核酸 - 靶标探针双链体，洗脱解离的靶标探针和测试标签来测试。 在另一个实施方案中，通过将钩连接到未标记的探针上，而无需首先除去未共价连接的探针，从而可以通过捕捉钩来回收标记的靶探针而进行测试。 在这两种情况下，诊断探针和连续探针的存在是标签出现在测定中所必需的。 然后将上述方法应用于遗传疾病的检测。

    Method of nucleic acid extraction
    8.
    发明授权
    Method of nucleic acid extraction 失效
    核酸提取方法

    公开(公告)号：US5346999A

    公开(公告)日：1994-09-13

    申请号：US328471

    申请日：1989-03-24

    申请人： Guy R. Cathcart ,  Paul D. Grossman ,  P. Eric Mayrand ,  Eric S. Nordman ,  Norman M. Whiteley

    发明人： Guy R. Cathcart ,  Paul D. Grossman ,  P. Eric Mayrand ,  Eric S. Nordman ,  Norman M. Whiteley

    IPC分类号： B01J19/00 ,  C12M1/00 ,  C12N9/06 ,  C12N15/10 ,  C40B60/14 ,  C07H23/00

    CPC分类号： C12N9/0012 ,  B01J19/0046 ,  C12M47/06 ,  C12M47/12 ,  C12N15/1003 ,  B01J2219/00286 ,  B01J2219/00308 ,  B01J2219/00331 ,  B01J2219/00389 ,  B01J2219/00488 ,  B01J2219/00689 ,  B01J2219/00695 ,  C40B60/14 ,  Y10T436/25125 ,  Y10T436/25375

    摘要： An automated apparatus is provided which implements a new method of extracting and purifying nucleic acids from cells without the use of centrifugation. In the method, a lysate is created by treating the cells with proteinase K in the presence of a lysis buffer having a high concentration of a salt. The lysate is mixed with a phenol-based solvent system, thereby creating an emulsion. The emulsion is heated to promote phase separation. Similarly, the rate of phase separation is also enhanced by increasing the surface area of the emulsion. Once the phase separation is complete, the lower organic phase is removed and the upper aqueous phase is repeatedly extracted with the phenol-based solvent a preselected number of times, and is finally extracted using chloroform. The remaining aqueous phase is then dialyzed to further purify and concentrate the nucleic acid solution. Two preferred embodiments of apparatus are presented to accomplish this extraction.

    摘要翻译： 提供了一种自动化装置，其实现了从细胞中提取和纯化核酸而不使用离心的新方法。 在该方法中，通过在具有高浓度盐的裂解缓冲液存在下用蛋白酶K处理细胞产生裂解物。 将裂解物与苯酚基溶剂体系混合，从而产生乳液。 将乳液加热以促进相分离。 类似地，通过增加乳液的表面积也增加了相分离速率。 一旦相分离完成，除去较低的有机相，并用苯酚类溶剂预选次数反复提取上部水相，最后用氯仿萃取。 然后将剩余的水相透析以进一步净化并浓缩核酸溶液。 呈现装置的两个优选实施例以完成该提取。