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    Length determination of nucleic acid repeat sequences by discontinuous primer extension
    41.
    发明授权
    Length determination of nucleic acid repeat sequences by discontinuous primer extension 失效
    通过不连续引物延伸确定核酸重复序列的长度

    公开(公告)号：US5945284A

    公开(公告)日：1999-08-31

    申请号：US863437

    申请日：1997-05-27

    申请人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

    发明人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  C07H21/02 ,  C07H21/04

    CPC分类号： C12Q1/6869

    摘要： In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle. In a second aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

    摘要翻译： 在第一方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 分离靶 - 引物杂交体和未反应的第一引物延伸试剂; 使用第二引物延伸试剂进行第二引物延伸反应，其中第一或第二引物延伸试剂中的至少一个包括具有附着标记的标记的可延伸核苷酸; 将靶引物杂交体与未反应的第二引物延伸试剂分离; 测量标签产生的信号; 处理标签以使标签不可检测; 并重复上述步骤，直到信号基本上小于在先前循环中检测到的信号。 在第二方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 将靶引物杂交体与未反应的第一引物延伸试剂分离; 使用第二引物延伸试剂和引物终止试剂进行第二引物延伸反应，所述引物终止试剂包括具有附着标记的核苷酸终止子; 从未反应的第二引物延伸试剂和未反应的引物终止试剂中分离靶引物杂交体; 测量标签产生的信号; 并重复上述步骤直到检测到指示掺入核苷酸终止子的信号。 本发明还包括可用于实践上述方法的试剂盒。

    Self-quenching fluorescence probe
    42.
    发明授权
    Self-quenching fluorescence probe 失效
    自熄荧光探针

    公开(公告)号：US5723591A

    公开(公告)日：1998-03-03

    申请号：US559405

    申请日：1995-11-15

    申请人： Kenneth J. Livak ,  Susan J.A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

    发明人： Kenneth J. Livak ,  Susan J.A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

    IPC分类号： C12N15/09 ,  C07H21/00 ,  C12N15/00 ,  C12Q1/68 ,  C07H19/00 ,  C07H21/02 ,  C07H21/04

    CPC分类号： C12Q1/6818 ,  C12Q1/6834 ,  C12Q1/6837

    摘要： An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

    摘要翻译： 提供了一种寡核苷酸探针，其包括荧光报道分子和能够猝灭报道分子荧光的猝灭剂分子。 构建寡核苷酸探针，使得探针在未杂交的情况下以至少一个单链构象存在，其中猝灭剂分子足够接近报道分子以淬灭报道分子的荧光。 当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。 通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。 结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。 此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。

    Process for detecting polymorphisms on the basis of nucleotide differences
    43.
    发明授权
    Process for detecting polymorphisms on the basis of nucleotide differences 失效
    基于核苷酸差异检测多态性的方法

    公开(公告)号：US5126239A

    公开(公告)日：1992-06-30

    申请号：US494258

    申请日：1990-03-14

    申请人： Kenneth J. Livak ,  Jan A. Rafalski ,  Scott V. Tingey ,  John G. Williams

    发明人： Kenneth J. Livak ,  Jan A. Rafalski ,  Scott V. Tingey ,  John G. Williams

    IPC分类号： C12N15/09 ,  C12Q1/68

    CPC分类号： C12Q1/6858 ,  Y10T436/143333

    摘要： A process is provided for detecting polymorphisms on the basis of nucleotide differences in random segments of the nucleic acid by performing a primer extension reaction on the nucleic acids and comparing the extension reaction products. The random nucleic acid segment can be amplified by first dissociating the extension product from the template and contacting the dissociated extension product with a primer under conditions such that an amplification extension product is synthesized using the dissociated extension product as a template. Differences in the extension product are useful as markers in constructing genetic maps and as markers to distinguish or identify individuals.

    摘要翻译： 提供了通过对核酸进行引物延伸反应并比较延伸反应产物来检测基于核酸随机区段的核苷酸差异的多态性的方法。 可以通过首先将延伸产物从模板中解离并使解离的延伸产物与引物接触并在使用解离的延伸产物作为模板合成扩增产物的条件下扩增随机核酸片段。 扩展产物的差异可用作构建遗传图谱的标记和用于区分或识别个体的标记。

    Method of gene mapping
    44.
    发明授权
    Method of gene mapping 失效
    基因图谱法

    公开(公告)号：US5102785A

    公开(公告)日：1992-04-07

    申请号：US185741

    申请日：1988-04-25

    申请人： Kenneth J. Livak ,  Sydney Brenner

    发明人： Kenneth J. Livak ,  Sydney Brenner

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  G01N33/50

    CPC分类号： C12Q1/6869 ,  C12Q1/6813 ,  C12Q1/683 ,  Y10T436/143333

    摘要： The method described characterizes each DNA segment to be mapped by cleaving it to produce DNA fragments which are then end labeled with a reporter(s) specific to the end nucleotides of each fragment. The labeled fragments are again cleaved to produce short fragments which are separated according to size. The short fragments are analyzed as to report identify and size which is indicative of the character of each fragment. By derivatizing the cleaved ends of the primary cleaved fragments, the labeling may be delayed until the second cleavage. Prior to the labeling the derivatized fragments, all underivatized fragments are removed, the derivatized fragments being immobilized.

    DNA sequencing methods and detectors and systems for carrying out the same
    46.
    发明授权
    DNA sequencing methods and detectors and systems for carrying out the same 有权
    DNA测序方法和检测器和系统进行相同

    公开(公告)号：US08969090B2

    公开(公告)日：2015-03-03

    申请号：US12984269

    申请日：2011-01-04

    申请人： Hongye Sun ,  Eric S. Nordman ,  Mark F. Oldham ,  John R. O'Neill ,  Charles Connell ,  Umberto Ulmanella ,  Aldrich N. K. Lau ,  Theofilos Kotseroglou ,  Kenneth J. Livak

    发明人： Hongye Sun ,  Eric S. Nordman ,  Mark F. Oldham ,  John R. O'Neill ,  Charles Connell ,  Umberto Ulmanella ,  Aldrich N. K. Lau ,  Theofilos Kotseroglou ,  Kenneth J. Livak

    IPC分类号： G01N27/447 ,  G01N33/543 ,  C12Q1/68 ,  G01N27/414 ,  G01Q60/24 ,  C08G69/42 ,  B01L3/00

    CPC分类号： C12Q1/6869 ,  B01L3/502761 ,  G01N27/3278 ,  G01N27/414 ,  G01N27/4145 ,  G01N27/4146 ,  G01N33/5438 ,  Y10S977/924 ,  Y10T436/143333 ,  C12Q2565/631

    摘要： In some embodiments, an analyte detection system is provided that includes a nanochannel, an electrode arrangement, and a plurality of nanoFET devices disposed in the nanochannel. A plurality of nucleic acid base detection components can be used that include a plurality of nanopores, a plurality of nanochannels, a plurality of hybridization probes, combinations thereof, and the like. According to other embodiments of the present teachings, different coded molecules are hybridized to a target DNA molecule and used to detect the presence of various sequences along the target molecule. A kit including mixtures of coded molecules is also provided. In some embodiments, devices including nanochannels, nanopores, and the like, are used for manipulating movement of DNA molecules, for example, in preparation for a DNA sequencing detection. Nanopore structures and methods of making the same are also provided as are methods of nucleic acid sequencing using the nanopore structures. Surface-modified nanopores are provided as are methods of making them. In some embodiments, surfaced-modified nanopores for slowing the translocation of single stranded DNA (ssDNA) through the nanopore are provided, as are nanopores configured to detect each of a plurality of different bases on an ssDNA strand.

    摘要翻译： 在一些实施例中，提供了分析物检测系统，其包括纳米通道，电极布置以及设置在纳米通道中的多个纳米器件器件。 可以使用多个核酸碱基检测组分，其包括多个纳米孔，多个纳米通道，多个杂交探针及其组合等。 根据本教导的其他实施方案，将不同的编码分子与靶DNA分子杂交并用于检测沿着靶分子的各种序列的存在。 还提供了包含编码分子混合物的试剂盒。 在一些实施方案中，包括纳米通道，纳米孔等的装置用于操纵DNA分子的移动，例如用于DNA测序检测的准备。 还提供了制备其的纳米孔结构及其制备方法，使用纳米孔结构的核酸测序方法也是如此。 提供表面改性的纳米孔是制备它们的方法。 在一些实施方案中，提供了用于减缓通过纳米孔的单链DNA（ssDNA）易位的表面改性的纳米孔，以及配置成检测ssDNA链上的多个不同碱基中的每一个的纳米孔。

    Systems and methods for isolating nucleic acids
    48.
    发明授权
    Systems and methods for isolating nucleic acids 失效
    用于分离核酸的系统和方法

    公开(公告)号：US08568580B2

    公开(公告)日：2013-10-29

    申请号：US12179455

    申请日：2008-07-24

    申请人： Charles S. Vann ,  Maxim G. Brevnov ,  David W. Ruff ,  Kenneth J. Livak

    发明人： Charles S. Vann ,  Maxim G. Brevnov ,  David W. Ruff ,  Kenneth J. Livak

    IPC分类号： G01N27/453 ,  B01D61/42

    CPC分类号： C12N15/101 ,  G01N27/44782

    摘要： A system for collecting target nucleic acids from a sample can include at least one sample chamber configured to receive a sample containing target nucleic acids and other material, at least one collection chamber removably mountable relative to the at least one sample chamber and configured to collect target nucleic acids separated from the other material, a filter removably mountable relative to the at least one sample chamber and configured to be disposed between the at least one sample chamber and the at least one collection chamber when the at least one collection chamber is mounted relative to the at least one sample chamber. The system may further include a pair of electrodes configured to generate an electric field sufficient to cause target nucleic acids in the at least one sample chamber to migrate via electrophoresis from the at least one sample chamber through the filter into the at least one collection chamber, wherein the filter may be configured to permit passage of target nucleic acids and to block passage of material of a size larger than the target nucleic acids.

    摘要翻译： 用于从样品收集目标核酸的系统可以包括至少一个样品室，其被配置为接收含有靶核酸和其它材料的样品，至少一个收集室，其相对于至少一个样品室可拆卸地安装并且被配置成收集目标 与其他材料分离的核酸，相对于至少一个样品室可拆卸地安装的过滤器，并且被配置为当所述至少一个收集室相对于所述至少一个收集室安装时，被设置在所述至少一个样品室和所述至少一个收集室之间 所述至少一个样品室。 该系统可以进一步包括一对电极，其被配置为产生足以使所述至少一个样品室中的靶核酸经由电泳从所述至少一个样品室通过所述过滤器迁移到所述至少一个收集室中的电场， 其中所述过滤器可以被配置为允许靶核酸的通过并阻止尺寸大于所述靶核酸的材料的通过。

    MULTIPLEXED AMPLIFICATION OF SHORT NUCLEIC ACIDS
    49.
    发明申请
    MULTIPLEXED AMPLIFICATION OF SHORT NUCLEIC ACIDS 审中-公开
    短期核酸的多重放大

    公开(公告)号：US20130184171A1

    公开(公告)日：2013-07-18

    申请号：US13615057

    申请日：2012-09-13

    申请人： Kai Qin LAO ,  Kenneth J. Livak ,  Neil A. Straus

    发明人： Kai Qin LAO ,  Kenneth J. Livak ,  Neil A. Straus

    IPC分类号： C12N15/10

    CPC分类号： C12N15/1065 ,  C12Q1/6851 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2525/161 ,  C12Q2563/179 ,  C12Q2537/143 ,  C12Q2525/207

    摘要： The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.

    摘要翻译： 本教导提供用于逆转录和扩增小核酸如微RNA的方法，组合物和试剂盒。 通过使用zip编码的茎 - 环逆转录引物以及包含经翻译的正向引物的基于PCR的预扩增反应来提供高水平的复用。 下游解码PCR中的检测器探针可以利用茎环逆转录引物引入的zip码。 在一些实施方案中，通过循环逆转录反应来实现进一步的扩增。 本教导还提供可用于进行本文所述的逆转录和扩增反应的组合物和试剂盒。