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1. 发明申请

WO2019219905A1 DROPLET MANIPULATION DEVICE AND METHOD 审中-公开
公开(公告)号：WO2019219905A1
公开(公告)日：2019-11-21
申请号：PCT/EP2019/062791
申请日：2019-05-17
申请人： BASE4 INNOVATION LIMITED
发明人： DUNNING, Alexander , WAEBER, Andreas Michael
IPC分类号： B01L3/00
摘要： A microfluidic device for manipulating microdroplets is provided. It includes (a) a microfluidic space defined at least in part by juxtaposition of opposed containing walls and (b) disposed on or within at least one of the containing walls optoelectrowetting electrode locations arranged in a pathway characterised in that in at least one direction substantially parallel to that of the pathway a region of the microfluidic space continuously varies or may be caused to continuously vary in depth; for example by tapering or cantilevering a resilient part of one of the walls. The device and its associated method of use are suitable components for nucleic acid sequencers and biomedical analytical devices.

2. 发明申请

WO2018234446A1 MICROFLUIDIC ANALYTICAL DEVICE 审中-公开
公开(公告)号：WO2018234446A1
公开(公告)日：2018-12-27
申请号：PCT/EP2018/066574
申请日：2018-06-21
申请人： BASE4 INNOVATION LIMITED
发明人： ISAAC, Thomas Henry , CUNHA, Pedro , SHERIDAN, Eoin , LOVE, David , PALMER, Rebecca , KELLY, Douglas J. , PODD, Gareth
IPC分类号： B01L3/00 , C12Q1/6869
摘要： A device for investigating a nucleic acid analyte characterised by comprising: • a first zone comprising an attachment site to which the analyte is attached; a first pathway for causing a first fluid medium to flow over the attachment site thereby allowing the nucleic acid to be progressively pyrophosphorolysed into its constituent nucleoside triphosphates; a second pathway for removing the nucleoside triphosphates from around the attachment site and a means for creating in the second pathway a second medium comprised of aqueous microdroplets in a water-immiscible carrier; • a microdroplet manipulation zone for manipulating the microdroplets using optically-mediated electrowetting comprised of: • a first composite wall comprised of  a first transparent substrate  a first transparent conductor layer on the substrate having a thickness in the range 70 to 250nm;  a photoactive layer activated by electromagnetic radiation in the wavelength range 400-1000nm on the conductor layer having a thickness in the range 300-1000nm and  a first dielectric layer on the conductor layer having a thickness in the range 120 to 160nm; • a second composite wall comprised of  a second substrate;  a second conductor layer on the substrate having a thickness in the range 70 to 250nm and  optionally a second dielectric layer on the conductor layer having a thickness in the range 120 to 160nm;  wherein the exposed surfaces of the first and second dielectric layers are disposed less than 10µm apart to define a microfluidic space adapted to contain microdroplets; • an A/C source to provide a voltage across the first and second composite walls connecting the first and second conductor layers; • a source of first electromagnetic radiation having an energy higher than the bandgap of the photoexcitable layer adapted to impinge on the photoactive layer to induce corresponding ephemeral first electrowetting locations on the surface of the first dielectric layer; • means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer so as to vary the disposition of the ephemeral electrowetting locations thereby creating at least one first electrowetting pathway along which the microdroplets may be caused to move; • an detection zone disposed downstream of the microdroplet manipulation zone or integral therewith and • a fluorescence or Raman-scattering detection system comprising a second source of electromagnetic radiation adapted to impinge on the microdroplets in the detection zone and a detector for detecting fluorescence or Raman-scattering emitted therefrom.

3. 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。

4. 发明申请

WO2017216211A1 POLYNUCLEOTIDE SEPARATION METHOD 审中-公开
标题翻译：多核苷酸分离方法
公开(公告)号：WO2017216211A1
公开(公告)日：2017-12-21
申请号：PCT/EP2017/064509
申请日：2017-06-14
申请人： BASE4 INNOVATION LIMITED
发明人： DEAR, Paul
IPC分类号： C12Q1/68 , C12N15/10
摘要： Various method of isolating a modified polynucleotide having the formula A-P-B from a crude mixture of modified polynucleotides also including those having the formulae A-P-A and B- P-B, wherein P is a polynucleotide region and A and B are different modifying moieties or nucleotide sequences are provided. They are all characterised by the steps of (a) reacting the crude mixture concurrently or consecutively with beads of one type capable of binding to moiety A but not B, and beads of a second type capable of binding to moiety B but not A; the two types of bead differing in one or more of their sizes, densities, paramagnetic properties or optical properties to produce an intermediate product; (b) fractionating the intermediate product based on the properties of each type of bead and of pairs of different types of bead conjoined by A-P-B polynucleotides, such that only or predominantly conjoined pairs of the two different types of bead are retained and (c) optionally, releasing one or both beads from such conjoined pairs such that the polynucleotide may be recovered.
摘要翻译：从经修饰的多核苷酸的粗制混合物中分离具有式APB的修饰的多核苷酸的各种方法，所述修饰的多核苷酸还包括具有式APA和B-PB的那些，其中P是多核苷酸区域，并且A和B是不同的提供了修饰部分或核苷酸序列。它们都是通过以下步骤来表征的：（a）使粗混合物同时或连续地与能够结合部分A但不结合B的一种珠子和能结合部分B但不结合A的第二种珠子反应; 这两种类型的珠子在其尺寸，密度，顺磁特性或光学特性的一种或多种方面不同以产生中间产品; （b）基于每种珠类型和由APB多核苷酸连接的成对不同类型的珠子的性质，分离中间产物，使得仅保留或主要连接两种不同类型的珠子，并（c）任选地，从这样的结合对中释放一个或两个珠子，使得可以回收多核苷酸。

5. 发明申请

WO2019243577A1 SEQUENCING METHOD USING MODIFIED NUCLEOSIDE POLYPHOSPHATES 审中-公开
公开(公告)号：WO2019243577A1
公开(公告)日：2019-12-26
申请号：PCT/EP2019/066475
申请日：2019-06-21
申请人： BASE4 INNOVATION LIMITED
发明人： BELL, Neil
IPC分类号： C12Q1/6806 , C12Q1/6823
摘要： A method of sequencing a polynucleotide is provided. It is characterised by comprising the 5 steps of; (1) reacting the polynucleotide with a Group IA, Group IIA or ammonium salt of an acid having the formula Y-H wherein Y- corresponds to the general formula (X-O) 2 P(=B)-(Z-P(=B)(O-X)) n - wherein n is an integer from 1 to 4; each Z- is selected independently from -O-, -NH- or -CH 2 -; each B is independently either O or S; the X groups are independently selected from -H, -Na, -K, alkyl, alkenyl, or a heterocyclic group with the proviso that when both Z and B correspond to -O- and when n is 1 at least one X group is not H and a pyrophosphorolysis enzyme under conditions such that the polynucleotide is progressively pyrophosphorolysed into a corresponding stream of single modified nucleoside polyphosphate molecules each comprised of a polyphosphate unit of formula Y-Q' wherein Q' is comprised of phosphate and a nucleoside unit including one of the constituent nucleobases of the polynucleotide; (2) reacting each modified single nucleoside polyphosphate in the stream in the presence of a polymerase and optionally a ligase with a probe system comprised of one or more oligonucleotide probes each having a capture site complementary to that of one of the nucleobases of the polynucleotide and labelled with one or more fluorophores characteristic thereof and which in the probe's unused state are non-fluorescing to produce a corresponding stream of used probes; (3) reacting each used probe in the stream with a restriction endonuclease and/or an exonuclease to render the fluorophores fluorescing and (4) analysing the characteristic fluorescence from the fluorescing fluorophores and inferring therefrom the nucleobase associated with captured modified single nucleoside triphosphate.

6. 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.

7. 发明申请

WO2018234448A1 METHOD FOR INVESTIGATING MOLECULES SUCH AS NUCLEIC ACIDS 审中-公开
公开(公告)号：WO2018234448A1
公开(公告)日：2018-12-27
申请号：PCT/EP2018/066579
申请日：2018-06-21
申请人： BASE4 INNOVATION LIMITED
发明人： FRAYLING, Cameron Alexander , CUNHA, Pedro , ISAAC, Thomas Henry
IPC分类号： B01L3/00 , C12Q1/6869
摘要： A method for manipulating a microdroplet of a reaction medium in an immiscible carrier medium with a target molecule bound to a solid support for the purposes of effecting a chemical transformation is provided. It is characterised by the steps of (a) bringing the microdroplet into contact with the solid support under conditions where the microdroplet and solid support are caused to combine, (b) allowing the reaction medium to react with the target molecule and (c) thereafter exerting a force to induce the reaction medium to become detached from the solid support and reform a microdroplet in the carrier fluid. In one embodiment the solid support is a particle, bead or the like.

8. 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.

9. 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。

10. 发明申请

WO2017140839A1 IMPROVED DROPLET SEQUENCING APPARATUS AND METHOD 审中-公开
标题翻译：改进的滴流测序装置和方法
公开(公告)号：WO2017140839A1
公开(公告)日：2017-08-24
申请号：PCT/EP2017/053607
申请日：2017-02-17
申请人： BASE4 INNOVATION LIMITED
发明人： FRAYLNG, Cameron Alexander , ISAAC, Thomas , SOSNA, Maciej
IPC分类号： B01J19/00 , B01L3/00 , C12Q1/68 , B01L3/02
CPC分类号： C12Q1/6869 , B01J19/00 , B01L3/0268 , B01L2200/0668 , B01L2200/142 , B01L2300/0636 , B01L2300/0829 , C12Q2563/131 , C12Q2563/137 , C12Q2563/149 , C12Q2565/301 , C12Q2565/629
摘要： A first apparatus for sequencing a polynucleotide analyte is provided an apparatus for sequencing a polynucleotide analyte comprising; • a first zone for generating a flowing stream of single nucleotides by progressive pyrophosphorolysis of a molecule of the analyte attached to a particle and exposed to a flowing aqueous medium; • a second zone for generating a corresponding stream of aqueous droplets from the aqueous medium and the nucleotide stream and wherein at least some of the droplets contain a single nucleotide and • a third zone for storing and/or for interrogating each droplet to reveal a property characteristic of the single nucleotide it may contain; characterised in that the first zone includes at least one chemically-modified substrate adapted to bind temporarily to a particle having at least two different regions adapted to bind respectively to the substrate and to the analyte. Suitably the particles comprise at least one metal coated first region(s) and at least one second region(s) comprised of reactive sites to which the analyte can be attached. In one embodiment, the two regions each comprise hemispheres of a substantially spherical bead.
摘要翻译：提供了一种用于对多核苷酸分析物进行测序的设备，其包括： ·第一区，用于通过连接至颗粒并暴露于流动的含水介质的分析物分子的渐进焦磷酸分解产生单核苷酸流动流; ·用于从含水介质和核苷酸流产生相应的含水液滴流的第二区，并且其中至少一些液滴含有单个核苷酸和·第三区用于存储和/或询问每个液滴以显示性质它可能含有的单核苷酸的特征; 其特征在于所述第一区域包括至少一个化学修饰的基材，所述化学修饰的基材适于临时结合具有至少两个不同区域的颗粒，所述至少两个不同区域适于分别结合所述基材和所述分析物。合适地，所述颗粒包含至少一个金属涂覆的第一区域和至少一个第二区域，所述第二区域包含可与分析物连接的反应位点。在一个实施例中，两个区域各自包括基本上球形的珠子的半球。

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