BR112018067422A2 - método de sequenciamento de um ácido nucleico e sistema de sonda biológica multicomponente - Google Patents

método de sequenciamento de um ácido nucleico e sistema de sonda biológica multicomponente

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Publication number
BR112018067422A2
BR112018067422A2 BR112018067422A BR112018067422A BR112018067422A2 BR 112018067422 A2 BR112018067422 A2 BR 112018067422A2 BR 112018067422 A BR112018067422 A BR 112018067422A BR 112018067422 A BR112018067422 A BR 112018067422A BR 112018067422 A2 BR112018067422 A2 BR 112018067422A2
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BR
Brazil
Prior art keywords
oligonucleotide
probe
nucleic acid
site
cut
Prior art date
Application number
BR112018067422A
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English (en)
Inventor
Balmforth Barnaby
Original Assignee
Base4 Innovation Ltd
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Publication date
Application filed by Base4 Innovation Ltd filed Critical Base4 Innovation Ltd
Publication of BR112018067422A2 publication Critical patent/BR112018067422A2/pt

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/319—Exonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/331—Methylation site specific nuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/501—Ligase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/125—Modifications characterised by incorporating agents resulting in resistance to degradation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/131—Modifications characterised by incorporating a restriction site
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/301—Hairpin oligonucleotides
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/30—Oligonucleotides characterised by their secondary structure
    • C12Q2525/307—Circular oligonucleotides
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/159—Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/107—Alteration in the property of hybridised versus free label oligonucleotides
    • C—CHEMISTRY; METALLURGY
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/30—Detection characterised by liberation or release of label
    • C12Q2565/301—Pyrophosphate (PPi)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/629—Detection means characterised by use of a special device being a microfluidic device

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

um método de sequenciamento de um ácido nucleico caracterizado pelo fato de que compreende as etapas de (1) gerar uma corrente de nucleotídeos únicos por pirofosforólise progressiva do ácido nucleico; (2) produzir pelo menos uma sonda usada de oligonucleotídeo substancialmente de cadeia dupla por reação, na presença de uma polimerase e de uma ligase, de um dos nucleotídeos únicos com um sistema de sonda correspondente que compreende (a) um primeiro oligonucleotídeo de cadeia simples marcado com primeira e segunda regiões de tipos de elementos detectáveis característicos em um estado não detectável localizado respectivamente sobre os lados de extremidade x' e y' de uma terceira região compreendendo um elemento de local de reconhecimento de enzima de restrição incluindo o local de captura e um local de bloqueio de exonuclease no seu lado x' (em que x' é 3' e y' é 5' ou x' é 5' e y' é 3') e (b) segundo e terceiro oligonucleotídeos de cadeia simples capazes de hibridizar com as regiões complementares no primeiro oligonucleotídeo que flanqueia o local de captura; (2a) ou (i) tratar a sonda usada com uma endonuclease de restrição dependente de substituição convencional ou cortante para cortar a primeira cadeia de oligonucleotídeos no local de reconhecimento se e apenas se o nucleotídeo único capturado compreender uma nucleobase que é substituída ou (ii) tratar a sonda usada com uma endonuclease de restrição sensível à substituição convencional ou cortante, para cortar a primeira cadeia de oligonucleotídeos no local de reconhecimento se e somente se o nucleotídeo único capturado compreender uma nucleobase que é não-substituída; (3) digerir a primeira cadeia oligonucleotídica da sonda utilizada com uma enzima possuindo atividade exonucleolítica de cadeia dupla na direção x'-y' correspondente ao primeiro oligonucleotídeo para produzir elementos detectáveis derivados ou da primeira região, da segunda região, ou das primeira e segunda regiões em um estado detectável e um quarto oligonucleotídeo de cadeia simples que é pelo menos em parte o complemento de sequência do primeiro oligonucleotídeo; (4) reagir o quarto oligonucleotídeo com outro primeiro oligonucleotídeo para produzir um produto de oligonucleotídeo substancialmente de cadeia dupla correspondente à sonda usada; (5) repetir as etapas (2a), (3) e (4) em um ciclo e (6) detectar os elementos detectáveis liberados em cada iteração da etapa (3), em que se a endonuclease utilizada for do tipo convencional, o segundo ou o terceiro oligonucleotídeo inclui uma ligação direcionadora de endonucleólise em ou perto de sua extremidade x' ou y' respectivamente. sistemas de sonda biológica correspondentes também são divulgados.
BR112018067422A 2016-02-24 2017-02-24 método de sequenciamento de um ácido nucleico e sistema de sonda biológica multicomponente BR112018067422A2 (pt)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP16157229.2A EP3211092B1 (en) 2016-02-24 2016-02-24 Single nucleotide detection method
PCT/EP2017/054306 WO2017144653A1 (en) 2016-02-24 2017-02-24 Single nucleotide detection method

Publications (1)

Publication Number Publication Date
BR112018067422A2 true BR112018067422A2 (pt) 2019-07-16

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Country Status (9)

Country Link
US (1) US20190062816A1 (pt)
EP (1) EP3211092B1 (pt)
JP (1) JP2019511915A (pt)
KR (1) KR20180117167A (pt)
CN (1) CN108699592A (pt)
AU (1) AU2017222975A1 (pt)
BR (1) BR112018067422A2 (pt)
CA (1) CA3014379A1 (pt)
WO (1) WO2017144653A1 (pt)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111263818A (zh) * 2017-10-23 2020-06-09 贝斯4创新公司 单核苷酸分析方法及相关探针
ES2874753T3 (es) 2018-07-19 2021-11-05 Biofidelity Ltd Método mejorado de detección de secuencia de polinucleótidos
GB201919186D0 (en) 2019-12-23 2020-02-05 Biofidelity Ltd Simplified polynucleotide sequence detection method
BR112023021384A2 (pt) 2021-04-15 2023-12-19 Biofidelity Enriquecimento e detecção de ácidos nucleicos

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0682671A4 (en) 1993-02-01 1998-01-14 Seq Ltd METHOD AND DEVICES FOR SEQUENCING DNA.
WO2003080861A1 (en) 2002-03-22 2003-10-02 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Single molecule sequencing using phosphate labeled nucleotides
GB201217772D0 (en) 2012-10-04 2012-11-14 Base4 Innovation Ltd Sequencing method
GB201217770D0 (en) 2012-10-04 2012-11-14 Base4 Innovation Ltd Biological probes and the use thereof
GB201300957D0 (en) 2013-01-18 2013-03-06 Base4 Innovation Ltd Sequencing method
GB201306444D0 (en) 2013-04-09 2013-05-22 Base4 Innovation Ltd Single nucleotide detection method
GB201306445D0 (en) 2013-04-09 2013-05-22 Base4 Innovation Ltd Single nucleotide detection method
GB201402644D0 (en) * 2014-02-14 2014-04-02 Base4 Innovation Ltd Methylation detection method
GB201412977D0 (en) 2014-07-22 2014-09-03 Base4 Innovation Ltd Single nucleotide detection method

Also Published As

Publication number Publication date
CN108699592A (zh) 2018-10-23
WO2017144653A1 (en) 2017-08-31
CA3014379A1 (en) 2017-08-31
US20190062816A1 (en) 2019-02-28
JP2019511915A (ja) 2019-05-09
AU2017222975A1 (en) 2018-09-13
EP3211092A1 (en) 2017-08-30
EP3211092B1 (en) 2019-03-27
KR20180117167A (ko) 2018-10-26

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