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1. 发明授权

US6136172A Gel-forming insert for electrophoresis gels 失效
公开(公告)号：US6136172A
公开(公告)日：2000-10-24
申请号：US470024
申请日：1999-12-22
申请人： John A. Renfrew , Eric Steinbach , Stuart MacMillan
发明人： John A. Renfrew , Eric Steinbach , Stuart MacMillan
IPC分类号： G01N27/447 , G01N27/26
CPC分类号： G01N27/44704 , G01N27/44743
摘要： Electrophoresis gels are formed using a gel-forming insert having a beveled edge which results in loading sites having a beveled bottom surface. The gel forming insert can have a continuous beveled edge across the entire width of the gel, in which case a special loading insert is used which matches the bevel of the gel. Alternatively, the gel forming insert may be formed with a plurality of fingers with beveled ends, each finger defining a well in the gel. In one form of the gel-forming insert, the fingers are formed from a soft, flexible polymer such as silicone applied on a rigid support.

2. 发明授权

US6071726A Method, reagents and kit for diagnosis and targeted screening for p53 mutations 失效
标题翻译：用于诊断和靶向筛选p53突变的方法，试剂和试剂盒
公开(公告)号：US6071726A
公开(公告)日：2000-06-06
申请号：US765626
申请日：1996-12-27
申请人： Eleftherios Diamandis , James M. Dunn , John K. Stevens
发明人： Eleftherios Diamandis , James M. Dunn , John K. Stevens
IPC分类号： C12Q1/68 , C12P19/34 , C07H21/04 , G01N33/53
CPC分类号： C12Q1/6827 , C12Q1/6858 , C12Q1/6886 , C12Q1/686 , C12Q1/6869 , C12Q2600/16
摘要： Rapid and cost effective diagnosis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost:(a) immunoassays,(b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene. Preferably, the amplification primers are multiplexed so that more than one DNA fragment is amplified in a single vessel, using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal p53 gene; and(c) analysis of DNA from a patient sample by DNA sequencing of the p53 gene beginning with the sequencing of those regions most likely to harbor point mutations, and proceeding to sequence regions less likely to harbor point mutations.
摘要翻译： PCT No.PCT / US95 / 08605 Sec。 371日期1996年12月27日第 102（e）日期1996年12月27日PCT提交1995年7月7日PCT公布。公开号WO96 / 01909 日期1996年1月25日通过使用所选择的多种诊断工具，以提高每个工具的精度和成本的层次结构实现对患者样本的p53突变的快速和成本有效的诊断，其中每个工具基本上不检测到假阳性。可以包括在所选择的多个测试中的诊断测试包括：提高准确性和成本的顺序：（a）免疫测定，（b）使用与内含子区域互补的扩增引物定量扩增p53外显子来分析来自患者样品的DNA 侧翼于每个外显子，并检查每个扩增片段的长度或数量，用于相对于正常p53基因的核苷酸插入或缺失。优选地，扩增引物被多重化，使得在单个容器中扩增多于一个DNA片段，使用提供用于扩增正常p53基因的特异长度的基因片段的引物组; 和（c）通过对最可能存在点突变的那些区域的测序开始的p53基因的DNA测序从患者样品中分析DNA，并且进行到不太可能存在点突变的序列区域。

3. 发明授权

US6025139A Method for identification of mutations using ligation of multiple oligonucleotide probes 失效
标题翻译：使用多个寡核苷酸探针连接鉴定突变的方法
公开(公告)号：US6025139A
公开(公告)日：2000-02-15
申请号：US11821
申请日：1998-02-23
申请人： Thomas D. Yager , James M. Dunn
发明人： Thomas D. Yager , James M. Dunn
IPC分类号： C12N15/09 , C12Q1/68 , C07H21/02 , C07H21/04 , C12N15/00 , C12P19/34
CPC分类号： C12Q1/6827
摘要： A ligase-based assay which relies only upon knowledge of the wild-type sequence of a gene or gene fragment is used to detect all types of mutations, i.e., point mutations, insertions and deletions. The assay makes use of a set of oligonucleotide probes, which may be packaged in kit form, which hybridize in series along the length of the gene. The ligation of the probes together form a ligation product, the size of which is evaluated. When the gene or gene fragment being analyzed corresponds to the normal sequence and thus perfectly matches the probes, all of the probes in the set are ligated together, and the ligation produce has a certain resulting size. When a mutation appears in the gene, the hybridization of the probe overlapping the mutation is impaired, with the result that some or all of the ligation produce is of smaller size. By evaluating the size of the ligation product, both the existence of a mutation and its approx. position can be identified.
摘要翻译： PCT No.PCT / US96 / 14020 Sec。 371日期1998年2月23日 102（e）1998年2月23日PCT PCT 1996年8月28日PCT公布。公开号WO97 / 08344 日期1997年3月6日仅依赖于基因或基因片段的野生型序列知识的基于连接酶的测定法用于检测所有类型的突变，即点突变，插入和缺失。该测定使用一组寡核苷酸探针，其可以以试剂盒形式包装，其沿着该基因的长度串联杂交。探针的连接形成连接产物，评价其尺寸。当分析的基因或基因片段对应于正常序列，因此完全匹配探针时，所有组中的所有探针连接在一起，连接产物具有一定的最终尺寸。当基因中出现突变时，与突变重叠的探针的杂交受损，结果是部分或全部连接产物的尺寸较小。通过评估连接产物的大小，都存在突变及其约。位置可以被识别。

4. 发明授权

US6024854A Method and apparatus for improving electrophoresis resolution 失效
标题翻译：改善电泳分辨率的方法和装置
公开(公告)号：US6024854A
公开(公告)日：2000-02-15
申请号：US96227
申请日：1998-06-11
申请人： Rodney D. Gilchrist
发明人： Rodney D. Gilchrist
IPC分类号： G01N27/447 , G01N27/26
CPC分类号： G01N27/44743
摘要： Electrophoretic separation of an analyte species in a sample is achieved with increased resolution by loading the sample onto a loading site of an electrophoresis gel, said loading site having a gel/buffer interface and then applying a focusing electric field to a first pair of electrodes to cause the analyte species to migrate to a narrow region disposed at or near the loading site to produce a focused sample. Then a separation electric field is applied to cause the analyte species in the focused sample to migrate through the electrophoresis gel and to be separated into bands. This method is preferably performed in an electrophoresis apparatus that is particularly adapted to practicing the method by virtue of the a pair of focusing electrodes which are positioned to cause migration of sample to a narrow region near the buffer/gel interface within the sample loading site of the gel. This actual location of this narrow region may be in a buffer region over the gel, or just within the gel near the loading site, for example within 500 microns of the loading site.
摘要翻译：通过将样品加载到电泳凝胶的加载位置上，通过提高分辨率来实现样品中分析物种的电泳分离，所述加载位点具有凝胶/缓冲液界面，然后将聚焦电场施加到第一对电极使分析物种迁移到设置在装载位置处或附近的狭窄区域以产生聚焦样品。然后施加分离电场以使聚焦样品中的分析物质迁移通过电泳凝胶并分离成条带。该方法优选在电泳装置中进行，该电泳装置特别适于通过一对聚焦电极来实施该方法，所述聚焦电极被定位成使得样品迁移到样品加载位置内的缓冲液/凝胶界面附近的窄区域凝胶。该窄区域的该实际位置可以在凝胶上方的缓冲区域中，或者仅在加载位置附近的凝胶内，例如在装载位点的500微米内。

5. 发明授权

US5888731A Method for identification of mutations using ligation of multiple oligonucleotide probes 失效
标题翻译：使用多个寡核苷酸探针连接鉴定突变的方法
公开(公告)号：US5888731A
公开(公告)日：1999-03-30
申请号：US590503
申请日：1996-01-24
申请人： Thomas D. Yager , James M. Dunn
发明人： Thomas D. Yager , James M. Dunn
IPC分类号： C12N15/09 , C12Q1/68 , C07H21/02 , C07H21/04
CPC分类号： C12Q1/6827
摘要： A ligase-based assay which relies only upon knowledge of the wild-type sequence of a gene or gene fragment is used to detect all types of mutations, i.e., point mutations, insertions and deletions. The assay makes use of a set of oligonucleotide probes, which may be packaged in kit form, which hybridize in series along the length of the gene. The ligation of the probes together form a ligation product, the size of which is evaluated. When the gene or gene fragment being analyzed corresponds to the normal sequence and thus perfectly matches the probes, all of the probes in the set are ligated together, and the ligation product has a certain resulting size. When a mutation appears in the gene, the hybridization of the probe overlapping the mutation is impaired, with the result that some or all of the ligation product is of smaller size. By evaluating the size of the ligation product, both the existence of a mutation and its approximate position can be identified.
摘要翻译：仅依赖于基因或基因片段的野生型序列的知识的基于连接酶的测定法用于检测所有类型的突变，即点突变，插入和缺失。该测定使用一组寡核苷酸探针，其可以以试剂盒形式包装，其沿着该基因的长度串联杂交。探针的连接形成连接产物，评价其尺寸。当分析的基因或基因片段对应于正常序列，从而完美匹配探针时，将所有组中的所有探针连接在一起，并且连接产物具有一定的最终尺寸。当基因中出现突变时，与突变重叠的探针的杂交受损，结果是部分或全部连接产物的尺寸较小。通过评估连接产物的大小，可以确定突变的存在及其近似位置。

6. 发明授权

US5853979A Method and system for DNA sequence determination and mutation detection with reference to a standard 失效
标题翻译：参考标准的DNA序列测定和突变检测的方法和系统
公开(公告)号：US5853979A
公开(公告)日：1998-12-29
申请号：US497202
申请日：1995-06-30
申请人： Ronald J. Green , Vrijmoed Chi , Rodney D. Gilchrist , Gregory Dee , John K. Stevens
发明人： Ronald J. Green , Vrijmoed Chi , Rodney D. Gilchrist , Gregory Dee , John K. Stevens
IPC分类号： C12Q1/68 , G01N27/447 , C12Q1/70 , G01N33/48 , G06K9/00
CPC分类号： G01N27/44721 , G01N27/44717 , G06F19/18 , G06F19/22 , Y10T436/143333
摘要： Normalization of experimental fragment patterns for nucleic acid polymers having putatively known sequences starts with obtaining at least one raw fragment pattern for the experimental sample. The raw fragment pattern represents the positions of a selected nucleic acid base within the polymer as a function of migration time or distance. This raw fragment pattern is conditioned using conventional baseline correction and noise reduction technique to yield a clean fragment pattern. The clean fragment pattern is then evaluated to determine one or more "normalization coefficients." These normalization coefficients reflect the displacement, stretching or shrinking, and rate of stretching or shrinking of the clean fragment, or segments thereof, which are necessary to obtain a suitably high degree of correlation between the clean fragment pattern and a standard fragment pattern which represents the positions of the selected nucleic acid base within a standard polymer actually having the known sequence as a function of migration time or distance. The normalization coefficients are then applied to the clean fragment pattern to produce a normalized fragment pattern which is used for base-calling in a conventional manner. This method may be implemented in an apparatus comprising a computer processor programmed to determine normalization coefficients for an experimental fragment pattern. This computer may be separate from the electrophoresis apparatus, or part of an integrated unit.
摘要翻译：具有推定已知序列的核酸聚合物的实验片段模式的归一化从获得实验样品的至少一个原始片段图案开始。原始片段图案表示聚合物内所选择的核酸碱基作为迁移时间或距离的函数的位置。使用常规的基线校正和降噪技术调节该原始片段模式以产生干净的片段模式。然后评估干净的片段模式以确定一个或多个“归一化系数”。这些归一化系数反映了清洁片段或其片段的位移，拉伸或缩小以及拉伸或收缩速率，这些片段或片段是清洁片段图案与标示片段图案之间适当高程度的相关性所必需的，实际上具有已知序列的标准聚合物内所选核酸碱基的位置作为迁移时间或距离的函数。然后将归一化系数应用于干净的片段模式以产生用于以常规方式的基本呼叫的归一化的片段模式。该方法可以在包括被编程为确定实验片段模式的归一化系数的计算机处理器的装置中实现。该计算机可以与电泳装置或集成单元的一部分分开。

7. 发明授权

US5776767A Virtual DNA sequencer 失效
标题翻译：虚拟DNA测序仪
公开(公告)号：US5776767A
公开(公告)日：1998-07-07
申请号：US570994
申请日：1995-12-12
申请人： John K. Stevens , James M. Dunn , Gregory Dee , James W. Cassidy
发明人： John K. Stevens , James M. Dunn , Gregory Dee , James W. Cassidy
IPC分类号： G01N27/447 , G06F19/00 , C12M3/00
CPC分类号： G01N27/44782
摘要： A virtual DNA sequencer combines a plurality of individual DNA sequencers. Samples of DNA or other nucleic acid from subjects are prepared and allocated in real time to particular lanes or sets of lanes in electrophoresis plates of the individual sequencers, with records kept of the allocations. The data resulting from the electrophoresis runs is collected and collated according to the identities of the subjects. The individual sequencers are networked, and each individual sequencer is preferably equipped with a data buffer large enough to accommodate all or substantially all of a data run, thus protecting the virtual sequencer from loss of valuable data in the event that the network is disrupted for some portion of the time of the data run. In this way, a plurality of sequencers is virtually the same as a single sequencer with a very large number of tracks each of which can run for a much longer sequencing run than an individual sequencer.
摘要翻译：虚拟DNA测序仪组合了多个单独的DNA测序仪。来自受试者的DNA或其他核酸的样品被制备并实时分配到各个定序器的电泳板中的特定泳道或泳道组，并保存有分配记录。根据受试者的身份收集和整理电泳运行产生的数据。各个顺控程序被联网，并且每个单独的定序器优选地配备有足够大的数据缓冲器，以容纳所有或基本上所有的数据运行，从而在网络被中断的情况下保护虚拟定序器免于丢失有价值的数据部分时间的数据运行。以这种方式，多个定序器与具有非常大数量的轨道的单个定序器几乎相同，每个轨道可以运行比单个定序器更长的排序运行。

8. 发明授权

US5776737A Method and composition for internal identification of samples 失效
标题翻译：样品内部鉴定方法和组成
公开(公告)号：US5776737A
公开(公告)日：1998-07-07
申请号：US361757
申请日：1994-12-22
申请人： James M. Dunn
发明人： James M. Dunn
IPC分类号： C12N15/10 , C12Q1/68 , C12P19/34 , C07H21/04 , C12Q1/70
CPC分类号： C12N15/1093 , C12N15/10 , C12Q1/68
摘要： Patient samples are identified by adding to the sample, preferably at the time it is taken, a plurality of identification oligonucleotides. The identification oligonucleotides are co-processed and sequenced at the same time as the sample. The resulting sequence analysis thus provides both the sequence of the region of interest in the sample DNA, and the sequence of the identification oligonucleotides which are used to confirm the identity of the patient. In an embodiment of the invention, a plurality of specially constructed identification oligonucleotides is used. Each identification oligonucleotide is constructed based upon a starting oligonucleotide and comprises (a) a primer site which is not homologous with DNA from the organism from which the sample is taken and which may be the same or different from the primer site of the other identification oligonucleotides; and (b) an identification region having the general formula -(M-N).sub.x - or -(M-N-N).sub.x - wherein N represents a nucleotide residue which is the same in the identification oligonucleotide as in the starting oligonucleotide, M represents a nucleotide residue which may be the same as or different from the starting oligonucleotide, with the proviso that at least one M residue in the identification region is different from the starting oligonucleotide, and x is an integer from 3 to 20. To ensure that there is no overlap between different sets of identification oligonucleotides, the identification oligonucleotides may be constructed such that the identification regions of each set are located at a different place along the starting oligonucleotide.
摘要翻译：通过向样品中加入优选的时间来鉴定患者样品多个鉴定寡核苷酸。鉴定寡核苷酸与样品同时进行共处理和测序。所得到的序列分析因此提供了样品DNA中感兴趣区域的序列和用于确认患者身份的鉴定寡核苷酸序列。在本发明的一个实施方案中，使用多个特别构建的鉴定寡核苷酸。基于起始寡核苷酸构建每个鉴定寡核苷酸，并且包含（a）与取自样品的生物体的DNA不同源的引物位点，并且可以与其他鉴定寡核苷酸的引物位点相同或不同 ; 和（b）具有通式 - （MN）x-或 - （MNN）x-的识别区，其中N表示与起始寡核苷酸相同的核苷酸残基，M表示核苷酸残基，可以与起始寡核苷酸相同或不同，条件是鉴定区中至少一个M残基与起始寡核苷酸不同，x是3至20的整数。为了确保不存在重叠不同组的鉴定寡核苷酸，可以构建鉴定寡核苷酸，使得每组的鉴定区位于起始寡核苷酸的不同位置。

9. 发明授权

US06110339A Nanofabricated separation matrix for analysis of biopolymers and methods of making and using same 失效
标题翻译：用于生物聚合物分析的纳米分离基质及其制备和使用方法
公开(公告)号：US06110339A
公开(公告)日：2000-08-29
申请号：US973932
申请日：1997-12-16
申请人： Thomas D. Yager , Paul Waterhouse , Alexandre M. Izmailov , Bruno Maruzzo , John K. Stevens , Marina T. Larson
发明人： Thomas D. Yager , Paul Waterhouse , Alexandre M. Izmailov , Bruno Maruzzo , John K. Stevens , Marina T. Larson
IPC分类号： G01N27/447
CPC分类号： G01N27/44704 , G01N27/44773 , G01N27/44791 , Y10T436/143333
摘要： Separation matrices useful in the formation of solid-state mm- to cm-scale devices for the rapid, high-resolution separation of single-stranded DNA ladder bands generated by the Sanger dideoxy- or Maxam/Gilbert chemical DNA sequencing procedures are formed from a solid support (1) having a plurality of posts (4) disposed on a first major surface thereof to form an obstacle course of posts (4) and pores (5). The posts are arranged in a regular X, Y array and are separated one from another by a distance of 100 nm or less, preferably 10 to 30 nm, and are optionally separated into lanes 2. The separation matrix can be manufactured by first forming a mold, preferably a reusable mold using lithography techniques. The mold is the reverse of the desired pattern of posts and pores of the obstacle course, and is used for casting the obstacle course. The cast obstacle course is then fused to a solid support and separated from the mold. Alternatively, the separation matrix can be formed from a polymer which undergoes specific and quantifiable swelling in the presence of a selected chemical compound. In this case, the matrix is cast on a mold in a conventional manner with a spacing between posts greater than the desired final spacing of 100 nm or less. For use, a buffer solution saturated with the specific chemical agent that controls swelling is added, causing the posts to swell to a defined amount to achieve the desired separation.
摘要翻译： PCT No.PCT / US96 / 09999 Sec。 371日期：1997年12月16日 102（e）日期1997年12月16日PCT提交1996年6月7日PCT公布。出版物WO96 / 42012 PCT 日期1996年12月27日用于形成固态mm至cm尺度装置的分离基质用于快速，高分辨率分离由Sanger双脱氧或Maxam / Gilbert化学DNA测序产生的单链DNA梯形带步骤由具有设置在其第一主表面上的多个柱（4）的固体支撑件（1）形成，以形成柱（4）和孔（5）的障碍物路线。柱以规则的X，Y阵列布置，并且彼此分离一个距离为100nm或更小，优选为10至30nm，并且任选地分离成通道2.分离基体可以通过首先形成模具，优选使用光刻技术的可重复使用的模具。模具与障碍物路线的柱和孔的期望图案相反，并且用于铸造障碍物路线。然后将铸造的障碍物道路融合到固体支撑物并与模具分离。或者，分离基质可以由在所选择的化合物存在下经历特异性和可定量溶胀的聚合物形成。在这种情况下，基体以常规方式在模具上铸造，柱之间的间距大于期望的最终间距为100nm或更小。为了使用，加入饱和了特定化学试剂的缓冲溶液以控制溶胀，导致柱膨胀至规定的量以达到所需的分离。

10. 发明授权

US6083699A Method for bi-directional sequencing of nucleic acid polymers 失效
标题翻译：核酸聚合物双向测序方法
公开(公告)号：US6083699A
公开(公告)日：2000-07-04
申请号：US9483
申请日：1998-01-20
申请人： James Leushner , May Hui , James M. Dunn , Marina T. Larson , Jean-Michel Lacroix , Robert Shipman
发明人： James Leushner , May Hui , James M. Dunn , Marina T. Larson , Jean-Michel Lacroix , Robert Shipman
IPC分类号： B01L7/00 , C12Q1/68 , C12Q1/70 , G01N35/00
CPC分类号： C12Q1/703 , B01L7/52 , B01L7/525 , C12Q1/6841 , C12Q1/6869 , C12Q1/6881 , C12Q1/689 , G01N2035/00237
摘要： A method is provided for simultaneously determining the positions of a selected nucleotide base in a target region of both strands of a denatured duplex nucleic acid polymer. The nucleic acid polymer is combined with a reactant mixture comprising first and second oligonucleotide primers, said primers binding to the sense and antisense strands, respectively, of the nucleic acid polymer at a location flanking the target region; a thermostable DNA polymerase; a chain-terminating nucleotide triphosphate complementary to the selected nucleotide base; and other reagents for synthesis of chain extension products to form a reaction mixture. This mixture is processed through a plurality of thermal cycles, each including at least a chain extension phase and a denaturation phase to produce chain extension products. These chain extension products are evaluated to determine the positions of the selected bases. The method of the invention differs from the prior art, because the first and second oligonucleotide primers are each labeled with different, spectroscopically-distinguishable fluorescent labels. The method therefore obtains information about both DNA strands simultaneously while providing improved sensitivity as a result of the non-linear increase in the amount of DNA which results from the production of additional templates molecules from unterminated fragments.
摘要翻译：提供了一种用于同时测定变性双链体核酸聚合物的两条链的靶区域中所选核苷酸碱基的位置的方法。核酸聚合物与包含第一和第二寡核苷酸引物的反应物混合物组合，所述引物分别与靶区域侧翼位置处的核酸聚合物的有义链和反义链结合; 热稳定DNA聚合酶; 与所选核苷酸碱基互补的链终止核苷酸三磷酸; 和其他用于合成链延伸产物的试剂形成反应混合物。该混合物通过多个热循环进行处理，每个热循环至少包含延伸相和变性相以产生链延伸产物。评估这些链延伸产物以确定所选碱基的位置。本发明的方法与现有技术不同，因为第一和第二寡核苷酸引物各自用不同的光谱可区分的荧光标记进行标记。因此，该方法同时获得关于两条DNA链的信息，同时由于从未终止的片段产生附加模板分子而导致的DNA量的非线性增加，提供了改善的灵敏度。

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