DK2576822T3 - Fremgangsmåde til dna-sekventering ved polymerisation - Google Patents

Fremgangsmåde til dna-sekventering ved polymerisation Download PDF

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Publication number
DK2576822T3
DK2576822T3 DK11722796.7T DK11722796T DK2576822T3 DK 2576822 T3 DK2576822 T3 DK 2576822T3 DK 11722796 T DK11722796 T DK 11722796T DK 2576822 T3 DK2576822 T3 DK 2576822T3
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Denmark
Prior art keywords
nucleic acid
double
stranded nucleic
acid molecule
polymerase
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DK11722796.7T
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English (en)
Inventor
David Bensimon
Vincent Croquette
Jean-François Allemand
Maria Manosas
Fang-Yuan Ding
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Centre Nat Rech Scient
Université Pierre Et Marie Curie Paris 6
Ecole Normale Supérieure
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Application filed by Centre Nat Rech Scient, Université Pierre Et Marie Curie Paris 6, Ecole Normale Supérieure filed Critical Centre Nat Rech Scient
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Publication of DK2576822T3 publication Critical patent/DK2576822T3/da

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    • 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
    • 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
    • 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
    • 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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • C12Q2521/319—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
    • 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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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (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)

Claims (12)

1. Fremgangsmåde til bestemmelse af en nukleinsyresekvens, hvilken fremgangsmåde omfatter trinnene: a) denaturering af et dobbeltstrenget nukleinsyremolekyle svarende til nukleinsyresekvensen, hvorved mindst en af baserne i en af den dobbeltstrengede nukleinsyres strenge er hæftet direkte eller indirekte til en bærer, og hvorved mindst en af baserne i den dobbeltstrengede nukleinsyres anden streng er hæftet til en bevægelig bærer; b) hybridisering af et enkeltstrenget nukleinsyremolekyle, "primeren", med det denaturerede dobbeltstrengede nukleinsyremolekyle; c) påføring af en spænding på den hybridiserede primer/det dobbeltstrengede nukleinsyremolekyle opnået i b); d) inkubering af den hybridiserede primer/det dobbeltstrengede nukleinsyremolekyle opnået i trin b) med en polymerase under betingelser, der vil føre til mindst én pause i replikation; og e) bestemmelse af pausens position ved replikation med hensyn til en ende af den dobbeltstrengede nukleinsyre, hvilken bestemmelse omfatter trinnene: • måling af afstanden (z) mellem de to ender af det dobbeltstrengede nukleinsyremolekyle, som er hæftet til bæreren; • måling af afstanden (Zhigh) mellem de to ender af det dobbeltstrengede nukleinsyremolekyle, som er hæftet til bæreren, når det dobbeltstrengede nukleinsyremolekyle er denatureret; og • sammenligning af z og zhjgh, og • bestemmelse af pausens position, hvorved det dobbeltstrengede nukleinsyremolekyle er en hårnålstruktur.
2. Fremgangsmåde ifølge krav 1, hvorved det dobbeltstrengede nukleinsyre denatureres i trin a) ved at bevæge bærerne væk.
3. Fremgangsmåde ifølge krav 2, hvorved en fysisk kraft over eller lig 15 pN, fortrinsvis over eller lig 17 pN, mere fortrinsvis over eller lig 18 pN, påføres det dobbeltstrengede molekyle ved at bevæge bærerne væk.
4. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved spændingen i trin c) ligger mellem 12 pN og 10 pN.
5. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved trinnene a) til e) gentages flere gange for at akkumulere målingerne og forhøje signal/støj-forholdet.
6. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved primeren forlænges af polymerasens aktivitet, og forlængelsesprocessen standses, inden polymerasen når det dobbeltstrengede nukleinsyremolekyles loop.
7. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, omfattende et yderligere trin med reducering af den fysiske kraft, der påføres det dobbeltstrengede nukleinsyremolekyle, til mindre end eller lig 5 pN.
8. Fremgangsmåde ifølge krav 7, hvorved polymerasens exonuklease-aktivitet aktiveres.
9. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, omfattende et yderligere trin med adskillelse af den nysyntetiserede streng.
10. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved trin a) til e) gentages.
11. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvorved polymerasen er et nukleotidselektivt enzym, og betingelserne i trin d) omfatter anvendelse af en reaktionsblanding, som modificerer hastigheden for enzymets processiv-bevægelse for et specificeret nukleotid.
12. Fremgangsmåde ifølge et hvilket som helst af kravene 1-10, hvorved betingelserne i trin d) omfatter anvendelse af en reaktionsblanding, der foruden deoxynukleotider omfattende dideoxynukleotider.
DK11722796.7T 2010-05-27 2011-05-26 Fremgangsmåde til dna-sekventering ved polymerisation DK2576822T3 (da)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP10305563A EP2390350A1 (en) 2010-05-27 2010-05-27 Method of DNA sequencing by polymerisation
US37762110P 2010-08-27 2010-08-27
PCT/EP2011/058664 WO2011147929A1 (en) 2010-05-27 2011-05-26 Method of dna sequencing by polymerisation

Publications (1)

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DK2576822T3 true DK2576822T3 (da) 2015-06-29

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US (2) US9493829B2 (da)
EP (2) EP2390350A1 (da)
JP (2) JP2013528380A (da)
KR (1) KR101848377B1 (da)
CN (1) CN103097551B (da)
AU (1) AU2011257227B2 (da)
CA (1) CA2800637C (da)
DK (1) DK2576822T3 (da)
ES (1) ES2539254T3 (da)
HK (1) HK1183912A1 (da)
IL (1) IL223256A (da)
WO (1) WO2011147929A1 (da)

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EP2390351A1 (en) 2010-05-27 2011-11-30 Centre National de la Recherche Scientifique (CNRS) Method of DNA sequencing by hybridisation
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WO2014113598A2 (en) 2013-01-16 2014-07-24 The Regents Of The University Of California Microfluidic devices to extract, concentrate and isolate molecules
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Publication number Publication date
KR101848377B1 (ko) 2018-04-13
IL223256A (en) 2017-10-31
KR20130123302A (ko) 2013-11-12
EP2576822B1 (en) 2015-03-18
WO2011147929A1 (en) 2011-12-01
CA2800637C (en) 2018-08-28
CA2800637A1 (en) 2011-12-01
US9493829B2 (en) 2016-11-15
HK1183912A1 (en) 2014-01-10
JP2013528380A (ja) 2013-07-11
US9738928B2 (en) 2017-08-22
CN103097551A (zh) 2013-05-08
CN103097551B (zh) 2016-01-20
JP6325028B2 (ja) 2018-05-16
EP2390350A1 (en) 2011-11-30
US20130171636A1 (en) 2013-07-04
AU2011257227B2 (en) 2015-07-09
EP2576822A1 (en) 2013-04-10
JP2016214250A (ja) 2016-12-22
IL223256A0 (en) 2013-02-03
ES2539254T3 (es) 2015-06-29
AU2011257227A1 (en) 2013-01-24
US20170073749A1 (en) 2017-03-16

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