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公开(公告)号：US10227638B2
公开(公告)日：2019-03-12
申请号：US15315482
申请日：2015-06-02
Applicant: BASE4 INNOVATION LTD
Inventor： Barnaby Balmforth
IPC: C12Q1/6869 , C12Q1/6823 , C12Q1/6827 , C12Q1/6811 , C12Q1/6858
Abstract: 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.
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公开(公告)号：US10144958B2
公开(公告)日：2018-12-04
申请号：US15118628
申请日：2015-02-13
Applicant: BASE4 INNOVATION LTD
IPC: C12Q1/68 , C07H21/00 , C12Q1/6827
Abstract: A method of determining whether a given single nucleotide is methylated or not methylated characterized by the steps of (a) contacting the single nucleotide with one or more hybridization probe types each of which in its unused form; (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; (c) treating the used probe with a methylation-dependent restriction or a methylation-sensitive restriction endonuclease to cleave adjacent the region resistant to exonucleolytic degradation; and thereafter (d) treating the product of step (c) 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 to determine if the single nucleotide is methylated or not.
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公开(公告)号：US20150275293A1
公开(公告)日：2015-10-01
申请号：US14433416
申请日：2013-10-04
Applicant: BASE4 INNOVATION LTD
Inventor： Cameron Alexander Frayling , Barnaby Balmforth , Bruno Flavio Nogueira de Sousa Soares , Thomas Henry Isaac , Boris Breiner , Alessandra Natale , Michele Amasio
IPC: C12Q1/68
CPC classification number: C12Q1/6876 , C12Q1/6823 , C12Q1/6869 , C12Q2521/301 , C12Q2521/319 , C12Q2525/301 , C12Q2563/107 , C12Q2565/1015 , C12Q2521/101 , C12Q2521/501
Abstract: 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.
Abstract translation: 公开了一种生物探针，其特征在于其包含单末端核苷酸区域，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。
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公开(公告)号：US20150247192A1
公开(公告)日：2015-09-03
申请号：US14433409
申请日：2013-10-04
Applicant: BASE4 INNOVATION LTD
Inventor： Cameron Alexander Frayling , Barnaby Balmforth , Bruno Flavio Nogueira de Sousa Soares , Thomas Henry Isaac , Boris Breiner , Alessandra Natale , Michele Amasio
IPC: C12Q1/68
CPC classification number: C12Q1/6874 , C12Q1/6869 , C12Q2521/101 , C12Q2521/301 , C12Q2521/319 , C12Q2521/501 , C12Q2525/301 , C12Q2563/159 , C12Q2565/1015 , C12Q2565/629
Abstract: 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.
Abstract translation: 公开了一种用于测序多核苷酸分析物的方法，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。 在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。
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公开(公告)号：US10000802B2
公开(公告)日：2018-06-19
申请号：US14682401
申请日：2015-04-09
Applicant: BASE4 INNOVATION LTD
Inventor： Cameron Alexander Frayling , Barnaby Balmforth , Bruno Flavio Nogueira de Sousa Soares , Thomas Henry Isaac , Boris Breiner , Alessandra Natale , Michele Amasio
IPC: C12Q1/68
CPC classification number: C12Q1/6874 , C12Q2563/159 , C12Q2565/629
Abstract: Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte characterised by the steps of: a. generating a stream of droplets at least some of which contain a single nucleotide and 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 (i) comprising a different detectable element in an undetectable state and (ii) being adapted to capture a different complimentary single nucleotide from which the analyte is constituted; c. causing the single nucleotide contained in the droplet to bind to its complimentary probe to create a used probe; and d. causing the detectable element to be released from the used probe in a detectable state. Typically the biological probe employed 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 essentially undetectable in the probe's unused state. In a most preferred embodiment the probe is labelled with multiple fluorophores and further comprises a restriction enzyme recognition site generated by the binding of the target single nucleotide to the single-stranded nucleotide region. Suitably, step c is carried out in the presence of a polymerase and ligase and step d in the presence of a restriction enzyme and an exonuclease. Typically the flow rate of the droplets is 100 to 2000 droplets per second.
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Patent Agency Ranking
公开(公告)号：US09546996B2
公开(公告)日：2017-01-17
申请号：US14413081
申请日：2013-07-09
Applicant: BASE4 INNOVATION LTD
Inventor： Bruno Flavio Nogueira de Sousa Soares , Cameron Alexander Frayling , Barnaby Balmforth , Michele Amasio
Abstract: The present invention provides an apparatus for analyzing 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 characterized 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.
Abstract translation: 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适于使核酸分子通过其中的多个纳米孔; 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。
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公开(公告)号：US20140367259A1
公开(公告)日：2014-12-18
申请号：US14366175
申请日：2012-12-20
Applicant: BASE4 INNOVATION LTD
IPC: G01N33/487 , C12Q1/68 , G01J3/44 , G01N27/447 , G01N21/65
CPC classification number: G01N33/48721 , C12Q1/6869 , C12Q1/689 , G01J3/4406 , G01N21/6428 , G01N21/648 , G01N21/658 , G01N27/447 , G01N2021/6432 , G01N2021/6441 , G01N2201/069 , C12Q2565/631
Abstract: 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.
Abstract translation: 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），从而识别目标聚合物（20）。 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。
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公开(公告)号：US09771615B2
公开(公告)日：2017-09-26
申请号：US14433409
申请日：2013-10-04
Applicant: BASE4 INNOVATION LTD
Inventor： Cameron Alexander Frayling , Barnaby Balmforth , Bruno Flavio Nogueira de Sousa Soares , Thomas Henry Isaac , Boris Breiner , Alessandra Natale , Michele Amasio
IPC: C12Q1/68
CPC classification number: C12Q1/6874 , C12Q1/6869 , C12Q2521/101 , C12Q2521/301 , C12Q2521/319 , C12Q2521/501 , C12Q2525/301 , C12Q2563/159 , C12Q2565/1015 , C12Q2565/629
Abstract: 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.
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公开(公告)号：US20150204840A1
公开(公告)日：2015-07-23
申请号：US14413081
申请日：2013-07-09
Applicant: BASE4 INNOVATION LTD
Inventor： Bruno Flavio Nogueira de Sousa Soares , Cameron Alexander Frayling , Barnaby Balmforth , Michele Amasio
IPC: G01N33/487
Abstract: 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.
Abstract translation: 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适于使核酸分子通过其中的多个纳米孔; 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。
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公开(公告)号：US10690689B2
公开(公告)日：2020-06-23
申请号：US15725646
申请日：2017-10-05
Inventor： Cameron Alexander Frayling , Barnaby Balmforth , Bruno Flavio Nogueira de Sousa Soares , Thomas Henry Isaac , Boris Breiner , Alessandra Natale , Michele Amasio , Paul Dear
IPC: G01N35/08 , C12Q1/6869 , B01L3/00
Abstract: A microfluidic sequencing device in which a stream of microdroplets at least some of which contain a single nucleotide base are made to undergo reaction with a capture system to capture and detect an ordered sequence of single nucleotide bases generated by progressive pyrophosphorolysis.
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