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1. 发明申请

WO2018046521A1 SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES 审中-公开
标题翻译：单核苷酸检测方法及相关探针
公开(公告)号：WO2018046521A1
公开(公告)日：2018-03-15
申请号：PCT/EP2017/072308
申请日：2017-09-06
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander
IPC分类号： C12Q1/68
摘要： A method of sequencing a nucleic acid is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one substantially double-stranded primary 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 primary probe comprising (a) a first single- stranded oligonucleotide including a restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the first oligonucleotide flanking regions; (3) nicking the first oligonucleotide strand of the used primary probe at the nicking-site with a nicking restriction endonuclease to create separate first oligonucleotide components; (4) separating the first oligonucleotide components generated in step (3) from the complementary strand of the used probe; (5) producing at least one substantially double-stranded secondary used probe by reacting, in the presence of a ligase, at least one of the separated first oligonucleotide components with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide bearing fluorophores in a substantially undetectable state and optionally (d) a fifth oligonucleotide at least in part complementary to the fourth oligonucleotide; (6) digesting the used secondary probe with an enzyme having double-stranded exonucleolytic activity to yield the fluorophores in a detectable state and a single-stranded sixth oligonucleotide which is at least in part the sequence complement of the fourth oligonucleotide and (7) detecting the fluorophores released in step (6). The method is advantageously carried out in microdroplets. Corresponding biological probe systems comprised of the primary and secondary probes are also described.
摘要翻译：提供了测序核酸的方法。其特征在于以下步骤：（1）通过核酸的渐进式酶消化产生单核苷三磷酸酯流; （2）通过在聚合酶和连接酶存在下，将至少一种单核苷三磷酸与相应的初级探针反应，产生至少一种基本上双链的初级寡核苷酸探针，所述初级探针包含（a）第一单链包括限制性内切核酸酶切口位点的寡核苷酸，捕获单个核苷三磷酸的单个核苷酸捕获位点和位于捕获位点两侧并置的寡核苷酸侧翼区域，和（b）能够与第一个寡核苷酸侧翼杂交的第二个和第三个单链寡核苷酸区域; （3）用切口限制性内切核酸酶在切口位点切割所用初级探针的第一条寡核苷酸链，以产生单独的第一寡核苷酸组分; （4）将步骤（3）中产生的第一寡核苷酸组分与所用探针的互补链分开; （5）通过在连接酶存在下，使至少一种分离的第一寡核苷酸组分与相应的第二探针反应，产生至少一种基本上双链的二次使用的探针，所述相应的第二探针包含（c）带有荧光团的互补第四寡核苷酸，基本上不可检测的状态，并且任选地（d）至少部分与第四寡核苷酸互补的第五寡核苷酸; （6）用具有双链核酸外切核酸活性的酶消化所用的第二探针以产生处于可检测状态的荧光团和至少部分为第四寡核苷酸的序列互补物的单链第六寡核苷酸，以及（7）检测在步骤（6）中释放荧光团。该方法有利地以微滴进行。还描述了由初级和次级探针组成的相应生物探针系统。

2. 发明申请

WO2018054964A1 SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES 审中-公开
标题翻译：单核苷酸检测方法及相关探针
公开(公告)号：WO2018054964A1
公开(公告)日：2018-03-29
申请号：PCT/EP2017/073759
申请日：2017-09-20
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander
IPC分类号： C12Q1/68
摘要： A method of sequencing a nucleic acid is provided. It is characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one substantially double-stranded primary 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 primary probe comprising (a) a first single- stranded oligonucleotide including a first restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide flanking regions juxtaposed either side of the capture site and (b) second and third single-stranded oligonucleotides capable of hybridising to the first oligonucleotide flanking regions; (3) nicking the first oligonucleotide strand of the used primary probe at the first nicking-site with a first nicking restriction endonuclease to create separate first oligonucleotide components; (4) separating the first oligonucleotide components generated in step (3) from the complementary strand of the used probe; (5) producing at least one substantially double-stranded secondary used probe by reacting, in the presence of a ligase, at least one of the separated first oligonucleotide components with a corresponding secondary probe comprising (c) a complementary fourth oligonucleotide including a second restriction endonuclease nicking-site and bearing fluorophores in a substantially undetectable state and optionally (d) a single-stranded fifth oligonucleotide at least in part complementary to the fourth oligonucleotide; (6) nicking the fourth oligonucleotide strand of the used secondary probe with a second nicking restriction endonuclease to create separate fourth oligonucleotide components at least some of which bear fluorophores in a detectable state and a single-stranded sixth oligonucleotide which is at least in part the sequence complement of the fourth oligonucleotide and (7) detecting the fluorophores released in step (6).
摘要翻译：提供了测序核酸的方法。其特征在于以下步骤：（1）通过核酸的渐进式酶消化产生单核苷三磷酸酯流; （2）通过在聚合酶和连接酶存在下，将至少一种单核苷三磷酸与相应的初级探针反应，产生至少一种基本上双链的初级寡核苷酸探针，所述初级探针包含（a）第一单链包括第一限制性内切核酸酶切口位点的寡核苷酸，用于捕获单个核苷三磷酸的单个核苷酸捕获位点和位于捕获位点两侧并置的寡核苷酸侧翼区域，和（b）能够与第一寡核苷酸杂交的第二和第三单链寡核苷酸侧翼区域; （3）用第一切口限制性内切核酸酶在第一切口位点切割所用初级探针的第一寡核苷酸链以产生单独的第一寡核苷酸组分; （4）将步骤（3）中产生的第一寡核苷酸组分与所用探针的互补链分开; （5）通过在连接酶存在下将至少一种分离的第一寡核苷酸组分与相应的第二探针反应，产生至少一种基本上双链的二次使用的探针，所述第二探针包含（c）互补的第四寡核苷酸，所述互补的第四寡核苷酸包含第二限制（d）至少部分与第四寡核苷酸互补的单链第五寡核苷酸;和（d）与第四寡核苷酸至少部分互补的单链第五寡核苷酸; （6）用第二切口限制性内切核酸酶切割所用的第二探针的第四寡核苷酸链以产生单独的第四寡核苷酸组分，所述第四寡核苷酸组分至少部分带有处于可检测状态的荧光团和单链第六寡核苷酸，所述单链第六寡核苷酸至少部分（7）检测步骤（6）中释放的荧光团。

3. 发明申请

WO2017144653A1 SINGLE NUCLEOTIDE DETECTION METHOD 审中-公开
标题翻译：单核苷酸检测方法
公开(公告)号：WO2017144653A1
公开(公告)日：2017-08-31
申请号：PCT/EP2017/054306
申请日：2017-02-24
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6827 , C12Q2521/319 , C12Q2521/331 , C12Q2521/501 , C12Q2525/125 , C12Q2525/131 , C12Q2563/159 , C12Q2565/107 , C12Q2565/301 , C12Q2565/629 , C12Q2525/307 , C12Q2525/301
摘要： A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleotides 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, one of the single nucleotides with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with first and second regions of characteristic detectable element types in an undetectable state located respectively on the X' and Y' end sides of a third region comprising a restriction enzyme recognition site element including the capture site and an exonuclease-blocking site on the X' side thereof (wherein either X' is 3' and Y' is 5' or X' is 5' and Y' is 3') and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide flanking the capture site; (2a) either (i) treating the used probe with a conventional or nicking substitution-dependent restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is substituted or (ii) treating the used probe with a conventional or nicking substitution-sensitive restriction endonuclease to cut the first oligonucleotide strand at the recognition site if and only if the single nucleotide captured comprises a nucleobase which is unsubstituted; (3) digesting the first oligonucleotide strand of the used probe with an enzyme having double-stranded exonucleolytic activity in the X'-Y' direction corresponding to the first oligonucleotide to yield detectable elements derived from either the first region, the second region, or the first and second regions 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 (2a), (3) and (4) in a cycle and (6) detecting the detectable elements released in each iteration of step (3) wherein if the endonuclease employed is of the conventional type the second or third oligonucleotide includes an endonucleolysis directing linkage at or close to its X' or Y' end respectively. Corresponding biological probe systems are also disclosed.
摘要翻译：对核酸进行测序的方法，其特征在于以下步骤：（1）通过核酸的进行性焦磷酸分解产生单核苷酸流; （2）通过在聚合酶和连接酶存在下，使单核苷酸中的一个与相应的探针系统反应，产生至少一个基本上双链寡核苷酸的探针，所述探针系统包含（a）用第一标记的第一单链寡核苷酸以及分别位于包含限制酶识别位点元件的第三区域的X'和Y'端侧的处于不可检测状态的特征可检测元件类型的第二区域，所述限制酶识别位点元件在其X'侧包含捕获位点和外切核酸酶阻断位点（其中X'为3'且Y'为5'或X'为5'且Y'为3'）和（b）第二和第三单链寡核苷酸，所述第二和第三单链寡核苷酸能够与第一寡核苷酸上的互补区杂交捕获地点; （2a）（i）当且仅当所捕获的单个核苷酸包含被取代的核碱基或（ii）处理（i）用常规或切口取代依赖性限制性内切核酸酶处理用过的探针以在识别位点切割第一寡核苷酸链时当且仅当捕获的单个核苷酸包含未被取代的核碱基时，所用的具有常规或切口取代敏感性限制性内切核酸酶的探针在识别位点切割第一寡核苷酸链; （3）用与第一寡核苷酸对应的X'-Y'方向的具有双链核酸外切酶活性的酶消化所用探针的第一寡核苷酸链，以产生源自第一区域，第二区域或第二区域的可检测元件处于可检测状态的第一和第二区域以及至少部分为第一寡核苷酸的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一个第一寡核苷酸反应以产生对应于所用探针的基本上双链的寡核苷酸产物; （5）在一个循环中重复步骤（2a），（3）和（4），和（6）检测在步骤（3）的每次重复中释放的可检测元素，其中如果使用的核酸内切酶是常规类型，则第二或第三寡核苷酸包括分别在其X'或Y'端或其附近的核苷酸内切引导连接。相应的生物探针系统也被公开。

4. 发明申请

WO2016116757A1 IMPROVED DROPLET SEQUENCING APPARATUS AND METHOD 审中-公开
标题翻译：改进的滴液测序装置和方法
公开(公告)号：WO2016116757A1
公开(公告)日：2016-07-28
申请号：PCT/GB2016/050130
申请日：2016-01-21
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander , ISAAC, Thomas Henry
IPC分类号： B01L3/00 , B01L3/02
CPC分类号： B01L3/502784 , B01F3/0807 , B01F13/0062 , B01L3/0268 , B01L3/502761 , B01L3/5088 , B01L2300/0819 , C12Q1/6869 , C12Q2563/159 , C12Q2565/629 , C12Q2563/149 , C12Q2565/301 , C12Q2565/518
摘要： An apparatus for sequencing a polynucleotide analyte is provided and comprises; • a first zone in which a stream of single nucleotides is generated by progressive digestion of a molecule of the analyte attached to a particle located therein and exposed to a flowing aqueous medium; • a second zone in which a corresponding stream of aqueous droplets is generated from the aqueous medium and the nucleotide stream and wherein at least some of the droplets contain a single nucleotide and • a third zone in which each droplet is stored and/or interrogated to reveal a property characteristic of the single nucleotide it may contain; characterised in that the first zone comprises a microfluidic channel through which the aqueous medium flows and the location comprises a hollow seating in a wall thereof to which suction can be applied and into which the particle can be close-fitted.
摘要翻译：提供了一种用于测序多核苷酸分析物的装置，包括： •第一区，其中通过逐渐消化附着于位于其中并暴露于流动水性介质的颗粒的分析物的分子产生单个核苷酸流; •第二区，其中从水性介质和核苷酸流产生相应的水滴流，并且其中至少一些液滴含有单个核苷酸和第三区，其中每个液滴被储存和/或询问到揭示其可能含有的单核苷酸的特性; 其特征在于，所述第一区域包括微流体通道，所述水性介质流过所述微流体通道，并且所述位置包括位于其壁中的中空座部，所述空腔可以被施加到所述空腔，所述颗粒可以紧密配合到所述壁中。

5. 发明申请

WO2019081483A1 SINGLE NUCLEOTIDE ANALYTICAL METHOD AND ASSOCIATED PROBES 审中-公开
公开(公告)号：WO2019081483A1
公开(公告)日：2019-05-02
申请号：PCT/EP2018/078991
申请日：2018-10-23
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander
IPC分类号： C12Q1/6823
CPC分类号： C12Q1/6823 , C12Q2521/101 , C12Q2521/301 , C12Q2521/319 , C12Q2525/117 , C12Q2525/125 , C12Q2525/307 , C12Q2563/159 , C12Q2537/149 , C12Q2563/107 , C12Q2537/164
摘要： A method of detecting whether the nucleobase of a given nucleoside triphosphate molecule in an analyte does or does not include a given chemical or structural modification characterised by the steps of (1) reacting the analyte in the presence of a polymerase with a biological probe comprised of (a) a pair of single-stranded first oligonucleotides each comprising an exonuclease blocking-site; at least one restriction endonuclease recognition-site located on the 3' side of the 10 blocking-site; a single nucleotide capture-site located within the recognition-site and respectively first and second fluorophore(s) located on the 3' side of the recognition-site arranged so as to be substantially undetectable and (b) at least one second and optionally at least one third single- stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary flanking regions on the 3' and 5' sides of the capture-site of one, other or both 15 of the first oligonucleotides in the pair to create a used-probe duplex consisting of (b1) one or other of the first oligonucleotides in the pair and (b2) a component comprised of the second oligonucleotide, one or other of a modified or unmodified nucleotide derived from the nucleoside triphosphate molecule and optionally the third oligonucleotide wherein each duplex is comprised of one of the four possible (b1)/(b2) duplex permutations; (2) reacting the duplex produced in 20 step (1) with a restriction endonuclease system comprised of at least one restriction endonuclease adapted to selectively cleave the (b1) strand at the recognition-site to create depending on the presence or absence of the modification in the original nucleoside triphosphate molecule (i) only exonucleolytically-digestible first oligonucleotide elements bearing first fluorophore(s) or (ii) only exonucleolytically-digestible first oligonucleotide elements bearing 25 second fluorophore(s) or (iii) a mixture of exonucleolytically-digestible first oligonucleotide elements respectively bearing first and second fluorophore(s); (3) creating another used-probe duplex from the (b2) component and another first oligonucleotide in the pair and thereafter iterating steps (2) and (3); (4) digesting the first oligonucleotide elements with an enzyme having 5'-3' exonucleolytic activity to produce fluorophore(s) in a detectable state after either or both of 30 steps (2) and (3); and (5) detecting the fluorophore(s) released in step (4) and inferring from the nature of the fluorescence signal observed whether the original single nucleoside triphosphate molecule was modified or unmodified.

6. 发明申请

WO2018210823A1 SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES 审中-公开
公开(公告)号：WO2018210823A1
公开(公告)日：2018-11-22
申请号：PCT/EP2018/062545
申请日：2018-05-15
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander , DETHLEFSEN, Mark
IPC分类号： C12Q1/6869 , C12Q1/6823
摘要： A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5' side of the blocking-site and including a single nucleotide capture-site for capturing the single nucleoside triphosphate, and at least one fluorophore region located on the 5' side of the recognition-site arranged so as to render the fluorophore(s) quenched and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary regions on the first oligonucleotide flanking the 3' and 5' sides of the capture-site; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease to create a first oligonucleotide component bearing the fluorophores; (4) digesting the first oligonucleotide component with an enzyme having 3'-5' exonucleolytic activity to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4). A corresponding method for use with enzymes having 5'-3' exonucleolytic activity as well as new biological probes and a kit derived therefrom is also provided.

7. 发明申请

WO2018042028A1 SINGLE NUCLEOTIDE DETECTION METHOD AND ASSOCIATED PROBES 审中-公开
标题翻译：单核苷酸检测方法及相关探针
公开(公告)号：WO2018042028A1
公开(公告)日：2018-03-08
申请号：PCT/EP2017/072063
申请日：2017-09-04
申请人： BASE4 INNOVATION LIMITED
发明人： BALMFORTH, Barnaby , FRAYLING, Cameron Alexander
IPC分类号： C12Q1/68
摘要： 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 enzymatic digestion 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 biological probe comprising (a) a first single-stranded oligonucleotide including a restriction endonuclease nicking-site, a single nucleotide capture site for capturing the single nucleoside triphosphate and oligonucleotide regions juxtaposed either side of the nicking-site bearing respectively at least one fluorophore and at least one quencher so as to render the fluorophores quenched and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide either side of the capture site; (3) nicking the first oligonucleotide strand of the used probe at the nicking-site with a nicking restriction endonuclease to create separate first oligonucleotide components respectively bearing the fluorophores and the quenchers; (4) separating the first oligonucleotide components generated in step (3) from the complementary strand of the used probe and (5) detecting the fluorophores on the separated oligonucleotide component bearing them. The method is suitable for being performed in microdroplets. New biological probes and probe systems for use with the method are also described.
摘要翻译：提供了测序核酸如DNA或RNA的方法。其特征在于以下步骤：（1）通过核酸的渐进式酶消化产生单核苷三磷酸酯流; （2）通过在聚合酶和连接酶存在下，将至少一种单核苷三磷酸与相应的生物探针反应，产生至少一种基本上双链的寡核苷酸探针，所述探针包含（a）第一单链寡核苷酸包括限制性内切核酸酶切口位点，用于捕获单个核苷三磷酸的单个核苷酸捕获位点和分别位于切口位点的任一侧的带有至少一个荧光团和至少一个淬灭剂的寡核苷酸区域，以使得荧光团淬灭， b）能够与捕获位点任一侧的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）用切口限制性内切核酸酶在切口位点处切割所用探针的第一寡核苷酸链，以产生分别带有荧光团和猝灭剂的单独的第一寡核苷酸组分; （4）将步骤（3）中产生的第一寡核苷酸组分与所用探针的互补链分离，以及（5）检测分离的携带它们的寡核苷酸组分上的荧光团。该方法适用于在微滴中进行。还描述了用于该方法的新型生物探针和探针系统。

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