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1. 发明申请

WO2015185564A1 NUCLEOTIDE POLYMORPHISM DETECTION METHOD 审中-公开
标题翻译：核多糖多态性检测方法
公开(公告)号：WO2015185564A1
公开(公告)日：2015-12-10
申请号：PCT/EP2015/062280
申请日：2015-06-02
申请人： BASE4 INNOVATION LTD
发明人： BALMFORTH, Barnaby
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q1/6811 , C12Q1/6858 , C12Q2521/501 , C12Q2535/131 , C12Q2561/125
摘要： A method for characterising a DNA analyte comprised of one or more polynucleotide types characteristic of a site of nucleotide polymorphism each of which includes a target region having the formula -X-Y-Z- wherein X and Z are respectively first and second characteristic flanking oligonucleotide regions and Y is one of the variants constituting the site is provided. The method is characterised by the steps of; (a) reacting a single-stranded oligonucleotide including the target region derived from at least one of the polynucleotide types with a set of unused probes comprised of (i) a single-stranded first aptamer terminating at its 3' end in a sequence complementary to that of -X or –X-Y and (ii) one or more second single-stranded aptamers terminating at their 5' end in a sequence complementary to that of –Z-Y or –Z (as the case may be) and labelled with detectable elements which are in an undetectable state in the presence of a ligase to create a substantially double-stranded used probe comprised of the oligonucleotide, first aptamer and one of the second aptamers; (b) wholly or in part digesting the used probe with an exonuclease or polymerase exhibiting exonuclease activity in a 3' to 5' direction into its constituent single nucleotides at least one of which includes a detectable element now in a detectable state and (c) thereafter detecting the detection property associated with the now detectable element thereby identifying the nature of the Y variant and therefore the allele it gives rise to. A second mirror-image method is also disclosed. Also provided are vesicles in which the method can be carried out. The method is suitable for a range of diagnostic screening applications including the detection of mutant alleles associated with genetic disorders and cancer.
摘要翻译：一种用于表征DNA分析物的方法，所述DNA分析物由核苷酸多态性位点特征的一种或多种多核苷酸类型组成，每个核苷酸类型包括具有式-XYZ-的靶区域，其中X和Z分别是第一和第二特征侧翼寡核苷酸区域，Y是提供构成该网站的变体之一。该方法的特征在于以下步骤：（a）使包含衍生自至少一种多核苷酸类型的靶区域的单链寡核苷酸与一组未使用的探针反应，所述一组未使用的探针包含（i）在其3'末端以与SEQ ID NO：1的序列互补的序列终止的单链第一适配体 -X或-XY和（ii）一个或多个第二单链适配体以与-ZY或-Z（视具体情况而定）互补的序列在其5'末端终止，并用可检测元素标记在连接酶存在下处于不可检测的状态以产生由寡核苷酸，第一适体和第二适体之一组成的基本上双链的使用探针; （b）使用具有在3'至5'方向上显示外切核酸酶活性的外切核酸酶或聚合酶完全或部分地消化使用的探针到其组成单个核苷酸中，其中至少一个包括现在处于可检测状态的可检测元件，和（c）此后检测与现在可检测元件相关联的检测性质，从而确定Y变体的性质，并因此鉴定其产生的等位基因。还公开了第二镜像方法。还提供了可以进行该方法的囊泡。该方法适用于一系列诊断筛选应用，包括检测与遗传疾病和癌症相关的突变等位基因。

2. 发明申请

WO2016012789A1 SINGLE NUCLEOTIDE DETECTION METHOD 审中-公开
标题翻译：单核心检测方法
公开(公告)号：WO2016012789A1
公开(公告)日：2016-01-28
申请号：PCT/GB2015/052119
申请日：2015-07-22
申请人： BASE4 INNOVATION LTD
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6823 , C12Q2521/101 , C12Q2521/319 , C12Q2521/501 , C12Q2525/125 , C12Q2525/307 , C12Q2533/107 , C12Q2537/149 , C12Q2537/162 , C12Q2563/107 , C12Q2563/159 , C12Q2565/629
摘要： SINGLE NUCLEOTIDE DETECTION METHOD A method of sequencing a nucleic acid such as DNA or RNA is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid; (2) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase at least one of the single nucleoside triphosphates with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with characteristic detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (3) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the detectable elements in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (4) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (5) repeating steps (3) and (4) in a cycle and (6) detecting the characteristic detectable elements released in each iteration of step (3). Suitably the detectable elements are fluorophores. The method of the present invention generates a stronger fluorescence signal from a single nucleoside triphosphate than has been described previously. Suitable probe systems are also disclosed.
摘要翻译：单核苷酸检测方法提供了对核酸如DNA或RNA进行测序的方法。其特征在于以下步骤：（1）通过核酸的逐步焦磷酸裂解产生单核苷三磷酸的流; （2）通过在聚合酶和连接酶的存在下使至少一种单核苷三磷酸与相应的探针系统反应制备至少一种基本上双链寡核苷酸的探针，所述探针系统包含：（a）第一单链寡核苷酸，用特征可检测元素在不可检测的状态和（b）能够与第一寡核苷酸上的互补区杂交的第二和第三单链寡核苷酸; （3）用具有双链外切核酸酶活性的酶消化使用的探针以产生可检测状态的可检测元件和至少部分为第一寡核苷酸序列互补序列的单链第四寡核苷酸; （4）使第四寡核苷酸与另外的第一寡核苷酸反应以产生对应于所用探针的基本双链寡核苷酸产物; （5）在一个循环中重复步骤（3）和（4），（6）检测在步骤（3）的每个迭代中释放的特征可检测元素。适当地，可检测的元素是荧光团。本发明的方法从单个核苷三磷酸产生比之前描述的更强的荧光信号。还公开了合适的探针系统。

3. 发明申请

WO2015121675A1 METHYLATION DETECTION METHOD 审中-公开
标题翻译：甲基化检测方法
公开(公告)号：WO2015121675A1
公开(公告)日：2015-08-20
申请号：PCT/GB2015/050422
申请日：2015-02-13
申请人： BASE4 INNOVATION LTD
发明人： BALMFORTH, Barnaby , SILVA, Ana Luisa Bras dos Santos Ribeiro da
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q2521/319 , C12Q2521/331 , C12Q2521/501 , C12Q2525/125 , C12Q2525/301 , C12Q2565/107
摘要： A method of determining whether a given single nucleotide is methylated or not methylated characterised by the steps of (a) contacting the single nucleotide with one or more hybridisation probe types each of which in its unused form comprises (1) a first oligonucleotide to which is attached one or more first detectable elements in an essentially undetectable state and which further comprises (i) double- and single-stranded regions and (ii) a region resistant to exonucleolytic degradation attached to the end of the double-stranded region adjacent the single- stranded region and (2) a second single-stranded oligonucleotide to which is attached one or more second detectable elements also in an essentially undetectable state and which is adapted to be at least in part the nucleotide sequence compliment of the single-stranded region of the first oligonucleotide; (b) for the relevant probe type causing (i) the single nucleotide to bind to the region resistant to exonucleolytic degradation and the single-stranded region and (ii) the second oligonucleotide to bind to the single nucleotide and the single-stranded nucleotide region to create a substantially double-stranded used probe; either (c1) treating the used probe with a methylation-dependent restriction endonuclease under conditions whereby the used probe is cleaved adjacent the region resistant to exonucleolytic degradation into two double-stranded oligonucleotide products if the single nucleotide is methylated or (c2) treating the used probe with a methylation-sensitive restriction endonuclease under conditions whereby the used probe is cleaved adjacent the region resistant to exonucleolytic degradation into two double-stranded oligonucleotide products if the single nucleotide is not methylated; and thereafter either (d1) treating the product of step (c1) with an exonuclease or a polymerase exhibiting exonuclease activity to liberate either only first or both first and second detectable elements in a detectable state if the single nucleotide is methylated, or only the second detectable elements if not or (d2) treating the product of step (c2) with an exonuclease or a polymerase exhibiting exonuclease activity to liberate either only first or both first and second detectable elements in a detectable state if the single nucleotide is not methylated, or only the second detectable element if the single nucleotide is methylated.
摘要翻译：确定给定的单核苷酸是否被甲基化或未甲基化的方法，其特征在于以下步骤：（a）使单个核苷酸与一种或多种其未使用形式的杂交探针类型接触，所述杂交探针类型包含（1）第一寡核苷酸连接一个或多个第一可检测元件，其基本上不可检测的状态，并且还包含（i）双链和单链区域和（ii）与邻近单链区域的双链区域末端附着的外源核酸水解降解的区域，双链区域和（2）第二单链寡核苷酸，其连接有一个或多个第二可检测元件，其也处于基本上不可检测的状态，并且其适于至少部分地基于所述单链区域的核苷酸序列互补第一寡核苷酸; （b）对于相关探针类型，导致（i）单核苷酸结合抗外切核酸分解降解区域和单链区域，和（ii）第二寡核苷酸结合单核苷酸和单链核苷酸区域以产生基本上双链的探针; （c1）在甲基化依赖性限制性内切核酸酶处理所使用的探针的条件下，如果单核苷酸被甲基化，或（c2）处理所使用的（c2）处理所用的探针在邻近外切核酸分解降解的区域裂解成两个双链寡核苷酸产物在甲基化敏感性限制性内切核酸酶的探针条件下，如果单个核苷酸未被甲基化，则使用的探针在抗核外裂解降解的区域附近裂解成两条双链寡核苷酸产物; 然后（d1）用外切核酸酶或表达核酸外切酶活性的聚合酶处理步骤（c1）的产物，以仅在第一个或第二个可检测元件处于可检测状态时释放，如果单个核苷酸被甲基化，或只有第二个如果不是，或（d2）用外切核酸酶或表达核酸外切酶活性的聚合酶处理步骤（c2）的产物，如果单个核苷酸没有被甲基化，则只有第一个或第二个可检测元件在可检测的状态下释放，或者只有第二个可检测元素，如果单个核苷酸是甲基化的。

4. 发明申请

WO2014053854A1 SEQUENCING METHOD 审中-公开
标题翻译：序列方法
公开(公告)号：WO2014053854A1
公开(公告)日：2014-04-10
申请号：PCT/GB2013/052595
申请日：2013-10-04
申请人： BASE4 INNOVATION LTD
发明人： FRAYLING, Cameron Alexander , BALMFORTH, Barnaby , SOARES, Bruno Flavio Nogueira de Sousa , ISAAC, Thomas Henry , BREINER, Boris , NATALE, Alessandra , AMASIO, Michele
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , C12Q1/6869 , C12Q2521/101 , C12Q2521/301 , C12Q2521/319 , C12Q2521/501 , C12Q2525/301 , C12Q2563/159 , C12Q2565/1015 , C12Q2565/629
摘要： Disclosed is a method for sequencing a polynucleotide analyte comprising: • a. generating a stream of droplets containing a single nucleotide wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; • b. introducing into each droplet a plurality of biological probe types each type comprising a different label in an undetectable state and being adapted to capture a different single nucleotide; • c. causing the single nucleotide contained in the droplet to bind to its complementary probe and • d. causing the label to be released from the probe that has bound the nucleotide in a detectable state. The probe is a dumbbell shaped probe comprising fluorescent donor and quencher labels and a single nucleotide gap. After gap repair by a polymerase and a ligase, a restriction enzyme recognition site is cleaved by a restriction enzyme, followed by exonuclease digestion to release the labels.
摘要翻译：公开了一种用于对多核苷酸分析物进行测序的方法，包括：a。产生含有单个核苷酸的液滴流，其中液滴流中单个核苷酸的顺序对应于分析物中的核苷酸序列; •b。向每个液滴中引入多种生物探针类型，每种类型包括不可检测状态的不同标记，并适于捕获不同的单个核苷酸; • C。导致液滴中含有的单核苷酸与其互补探针结合，导致标签从已探测状态的核苷酸结合的探针中释放出来。探针是包含荧光供体和猝灭标记和单核苷酸间隙的哑铃形探针。通过聚合酶和连接酶进行间隙修复后，限制酶识别位点被限制酶切割，随后进行核酸外切酶消化以释放标记。

5. 发明申请

WO2014053853A1 BIOLOGICAL PROBES AND THE USE THEREOF 审中-公开
标题翻译：生物探针及其用途
公开(公告)号：WO2014053853A1
公开(公告)日：2014-04-10
申请号：PCT/GB2013/052594
申请日：2013-10-04
申请人： BASE4 INNOVATION LTD
发明人： FRAYLING, Cameron Alexander , BALMFORTH, Barnaby , SOARES, Bruno Flavio Nogueira de Sousa , ISAAC, Thomas Henry , BREINER, Boris , NATALE, Alessandra , AMASIO, Michele
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6876 , C12Q1/6823 , C12Q1/6869 , C12Q2521/301 , C12Q2521/319 , C12Q2525/301 , C12Q2563/107 , C12Q2565/1015 , C12Q2521/101 , C12Q2521/501
摘要： Disclosed is a biological probe characterised in that it comprises a single-stranded nucleotide region the ends of which are attached to two different oligonucleotide regions wherein at least one of the oligonucleotide regions comprises detectable elements having a characteristic detection property and wherein the detectable elements are so arranged on the oligonucleotide region that the detectable property is less detectable than when the same number detectable elements are bound to a corresponding number of single nucleotides. The biological probe is especially useful for capturing single nucleotides or single-stranded nucleotides to create a used probe which can be degraded by means of a restriction enzyme and an exonuclease to generate single nucleotides carrying a detectable element in a form which can be detected. Typically the detectable elements are fluorophores and the corresponding characteristic fluorescence is rendered undetectable in the probe by for example the use of multiple adjacent fluorophores or mixtures of fluorophores and quenchers attached thereto. Preferably the single stranded nucleotide region is comprised of a single nucleotide whose associated nucleotide base is one of the characteristic of the nucleotides bases found in DNA or RNA.
摘要翻译：公开了一种生物探针，其特征在于其包含单链核苷酸区域，其末端连接到两个不同的寡核苷酸区域，其中至少一个寡核苷酸区域包含具有特征检测性质的可检测元件，并且其中可检测元素为排列在寡核苷酸区域上，当相同数量的可检测元素与相应数量的单个核苷酸结合时，可检测性质较不可检测。生物探针对于捕获单个核苷酸或单链核苷酸特别有用，以产生可以通过限制酶和外切核酸酶降解的用过的探针，以产生携带可检测的形式的可检测元件的单个核苷酸。通常，可检测的元素是荧光团，并且通过例如使用多个相邻的荧光团或与之相连的荧光团和猝灭剂的混合物，在探针中使得相应的特征荧光变得不可检测。优选地，单链核苷酸区域由其相关核苷酸碱基是在DNA或RNA中发现的核苷酸碱基的特征之一的单个核苷酸组成。

6. 发明申请

WO2014009704A1 IMPROVED SEQUENCING APPARATUS 审中-公开
标题翻译：改进的测序设备
公开(公告)号：WO2014009704A1
公开(公告)日：2014-01-16
申请号：PCT/GB2013/051801
申请日：2013-07-09
申请人： BASE4 INNOVATION LTD
发明人： SOARES, Bruno Flavio Nogueira de Sousa , FRAYLING, Cameron Alexander , BALMFORTH, Barnaby , AMASIO, Michele
IPC分类号： G01N33/487
CPC分类号： G01N33/48721 , H01Q1/243 , H01Q1/521 , H01Q9/16 , H01Q9/265 , H01Q21/28
摘要： The present invention provides an apparatus for analysing the sequence of nucleotides in a nucleic acid sample, said apparatus comprising a substrate and a plurality of nanopores provided therein suitable for the passage of nucleic acid molecules therethrough; at least one sample holding chamber disposed upstream of the inlet of said nanopores, at least one detection window juxtaposed within or downstream of the outlet of each nanopore adapted to detect a property characteristic of one or more detectable elements associated with the nucleic acid as each nucleic acid molecule passes therethrough and a detector adapted to generate a data stream characteristic of the various detection events occurring in the detection window characterised in that the apparatus further comprises a means located within the sample holding chamber adapted to increase the local concentration of the nucleic acid sample adjacent the inlet of the nanopores relative to the bulk concentration thereof.
摘要翻译：本发明提供了一种用于分析核酸样品中核苷酸序列的装置，所述装置包括底物和其中提供的多个纳米孔，其适于使核酸分子通过其中; 设置在所述纳米孔的入口上游的至少一个样品保持室，至少一个检测窗口，并置在每个纳米孔的出口的内部或下游，适合于检测与每个核酸相关的一个或多个可检测元件的特性，酸分子通过其中并且检测器适于产生特征在检测窗口中发生的各种检测事件的数据流，其特征在于，该设备还包括位于样品保持室内的装置，其适于增加核酸样品的局部浓度邻近纳米孔的入口相对于其体积浓度。

7. 发明申请

WO2013093483A1 A METHOD FOR IDENTIFYING A TARGET POLYMER 审中-公开
标题翻译：一种识别目标聚合物的方法
公开(公告)号：WO2013093483A1
公开(公告)日：2013-06-27
申请号：PCT/GB2012/053216
申请日：2012-12-20
申请人： BASE4 INNOVATION LTD
发明人： FRAYLING, Cameron Alexander , SOARES, Bruno Flavio Nogueira de Sousa , BALMFORTH, Barnaby
IPC分类号： G01N21/64 , G01N21/65 , C12Q1/68 , G01J3/44 , G01N33/487
CPC分类号： G01N33/48721 , C12Q1/6869 , C12Q1/689 , G01J3/4406 , G01N21/6428 , G01N21/648 , G01N21/658 , G01N27/447 , G01N2021/6432 , G01N2021/6441 , G01N2201/069 , C12Q2565/631
摘要： A method for identifying a target polymer (20) comprises translocating a target polymer (20) having detectable elements (22), such as fluorophores (22), through an analysing device (24) comprising a nanopore (28) having a detection window (40), wherein the analysing device (24) is capable of plasmon resonance to produce a localised electromagnetic field which defines the detection window (40) detecting the detectable elements (22) as they pass through the detection window (40) to produce a distribution profile of the detectable elements (22) along the target polymer (20) and identifying the target polymer (20) by comparing the distribution profile to a reference set of distribution profiles for known polymers. In a preferred embodiment the target polymer (20) is a nucleic acid and the detectable elements (22) are oligonucleotides complimentary to at least two adjacent nucleotides therein. Exemplified is the use of 6-mer oligonucleotides.
摘要翻译：用于鉴定目标聚合物（20）的方法包括将具有可检测元素（22）如荧光团（22）的目标聚合物（20）穿过包含具有检测窗口的纳米孔（28）的分析装置（24） 40），其中所述分析装置（24）能够进行等离子体共振以产生限定所述检测窗口（40）的局部电磁场，所述检测窗口（40）在通过所述检测窗口（40）时检测所述可检测元件（22）以产生分布通过将分布曲线与已知聚合物的分布曲线的参考组进行比较来识别目标聚合物（20）来识别目标聚合物（20）的可检测元件（22）的轮廓。在优选的实施方案中，靶聚合物（20）是核酸，并且可检测元件（22）是与其中至少两个相邻核苷酸互补的寡核苷酸。示例是使用6聚体寡核苷酸。

8. 发明申请

WO2014167324A1 SINGLE NUCLEOTIDE DETECTION METHOD 审中-公开
标题翻译：单核心检测方法
公开(公告)号：WO2014167324A1
公开(公告)日：2014-10-16
申请号：PCT/GB2014/051106
申请日：2014-04-09
申请人： BASE4 INNOVATION LTD , MEDICAL RESEARCH COUNCIL
发明人： FRAYLING, Cameron Alexander , BALMFORTH, Barnaby , SOARES, Bruno Flavio Nogueira de Sousa , ISAAC, Thomas Henry , BREINER, Boris , NATALE, Alessandra , AMASIO, Michele , DEAR, Paul
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q2521/319 , C12Q2531/113 , C12Q2537/157 , C12Q2565/629 , C12Q2521/101 , C12Q2521/501 , C12Q2525/301 , C12Q2565/1015 , C12Q2565/301
摘要： A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element.
摘要翻译：提供了确定多核苷酸分析物中核苷酸碱基序列的方法。其特征在于以下步骤：（1）从分析物产生单核苷酸碱基流; （2）通过使每个单核苷酸碱基与捕获系统反应来产生捕获的分子; （3）扩增至少部分所捕获的分子以产生特征为单核苷酸碱基的多个扩增子; （4）用具有特征可检测元素的相应探针标记扩增子，和（5）检测可检测元件的特性。

9. 发明申请

WO2014167323A1 SINGLE NUCLEOTIDE DETECTION METHOD 审中-公开
标题翻译：单核心检测方法
公开(公告)号：WO2014167323A1
公开(公告)日：2014-10-16
申请号：PCT/GB2014/051105
申请日：2014-04-09
申请人： BASE4 INNOVATION LTD , MEDICAL RESEARCH COUNCIL
发明人： FRAYLING, Cameron Alexander , BALMFORTH, Barnaby , SOARES, Bruno Flavio Nogueira de Sousa , ISAAC, Thomas Henry , BREINER, Boris , NATALE, Alessandra , AMASIO, Michele , DEAR, Paul
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , B01L3/502784 , G01N35/08 , C12Q2521/301 , C12Q2521/319 , C12Q2521/525 , C12Q2525/301 , C12Q2537/157 , C12Q2565/629 , C12Q2521/101 , C12Q2521/501 , C12Q2563/159 , C12Q2565/1015 , C12Q2565/301
摘要： A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.
摘要翻译：提供了确定多核苷酸分析物中核苷酸碱基序列的方法。其特征在于以下步骤：（1）通过焦磷酸分解从分析物产生单核苷酸碱基流; （2）通过使每个单核苷酸碱基与标记有不可检测状态的可检测元件的捕获系统反应来产生捕获的分子; （3）以可检测状态从每个捕获的分子中释放可检测元素，和（4）检测如此释放的可检测元件，并确定其中的核苷酸碱基序列。该方法可以有利地用于涉及使用微滴的定序器。

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