DK2768972T3 - Fremgangsmåder og sammensætninger til nukleinsyresekventering - Google Patents
Fremgangsmåder og sammensætninger til nukleinsyresekventering Download PDFInfo
- Publication number
- DK2768972T3 DK2768972T3 DK12766838.2T DK12766838T DK2768972T3 DK 2768972 T3 DK2768972 T3 DK 2768972T3 DK 12766838 T DK12766838 T DK 12766838T DK 2768972 T3 DK2768972 T3 DK 2768972T3
- Authority
- DK
- Denmark
- Prior art keywords
- nucleotide
- sequencing
- incorporation
- fluorescence
- fluorophore
- Prior art date
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- C—CHEMISTRY; METALLURGY
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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
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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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
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Claims (14)
1. Fluorescensbaseret fremgangsmåde til sekventering ved syntese (SBS) til bestemmelse af sekvensen af et polynukleotid, som omfatter påvisning i en sekventeringsreaktion af inkorporeringen af tre forskellige typer påviselige nukleotidkonjugater i et polynukleotid og bestemmelse af inkorporeringen af en fjerde type nukleotid baseret på påvisningsmønstret af de tre forskellige typer påviselige nukleotider i polynukleotidet til derved bestemmelse af sekvensen af et polynukleotid, hvor inkorporeringen af tre forskellige typer påviselige nukleotidkonjugater påvises fra en signaltilstand; og hvor et første nukleotidkonjugat, der er bundet til en første fluorofor, påvises i en første kanal; et andet nukleotidkonjugat, der er bundet til en anden fluorofor, påvises i en anden kanal; et tredje nukleotidkonjugat påvises i den første kanal og den anden kanal, hvor det tredje nukleotidkonjugat er en blanding af konjugater, der adskiller sig ved at være bundet til en tredje fluorofor, der påvises i den første kanal, eller en fjerde fluorofor, der påvises i den anden kanal; og hvor inkorporeringen af den fjerde type nukleotid bestemmes fra en mørk tilstand.
2. Fremgangsmåde ifølge krav 1, hvor de fire fluoroforer er forskellige fluorescerende dele.
3. Fremgangsmåde ifølge krav 1, hvor den første fluorofor er identisk med den tredje fluorofor, og den anden fluorofor er identisk med den fjerde fluorofor.
4. Fremgangsmåde ifølge krav 2 eller 3, hvor inkorporeringen af det Ijerde nukleotid bestemmes ved fluorescenspåvisningsmønstret af de fluorescerende dele, og hvor fluorescenspåvisningsmønstret bestemmes ved en første og en anden billeddannelseshændelse.
5. Fremgangsmåde ifølge krav 4, hvor den første billeddannelseshændelse påviser et fluorescensmønster, der er forskelligt fra fluorescensmønstret, der påvises ved den anden billeddannelseshændelse.
6. Fremgangsmåde ifølge krav 5, hvor inkorporeringen af et eller flere nukleotider bestemmes ved forskellen i fluorescensmønstret mellem den første og anden billeddannelseshændelse.
7. Fremgangsmåde ifølge krav 1, hvor den ene eller de flere typer nukleotidkonjugater yderligere omfatter en eller flere linkersekvenser, og hvor den ene eller de flere linkersekvenser omfatter en eller flere af en spaltbar linker og en spacerlinker, hvor den spaltbare linker omfatter en eller flere spaltbare koblingsgrupper valgt fra gruppen, der består af et disulfid, en diol, en diazo, en ester, en sulfon, et azid, en allyl og en silylether.
8. Fremgangsmåde ifølge krav 1, hvor nukleotiderne omfatter fire modificerede nukleotidtyper, hvor tre modificerede nukleotidtyper er konjugeret til en eller flere påvisningsdele og en eller flere linkere placeret mellem nukleotidet og den ene eller de flere påvisningsdele, og hvor en fjerde nukleotidtype mangler en påvisningsdel, hvor påvisningen omfatter a) påvisning af et inkorporeringsmønster af de modificerede nukleotider i en sekventeringsreaktion til derved indfangning af et første påviseligt mønster, b) tilsætning af en eller flere sammensætninger til sekventeringsreaktionen til derved ændring af det første påviselige mønster og c) påvisning af et andet påviseligt mønster, og hvor d) sekvensen af polynukleotidprøven bestemmes baseret på de påviselige mønstre.
9. Fremgangsmåde ifølge krav 8, hvor inkorporering af den fjerde nukleotidtype bestemmes ved hjælp af fluorescenspåvisningsmønstrene af de andre tre nukleotidtyper i både det første og andet påviselige mønster.
10. Fremgangsmåde ifølge krav 8, hvor den ene eller de flere linkere omfatter en eller flere af en spaltbar linker og en spacerlinker.
11. Fremgangsmåde ifølge krav 8, hvor den ene eller de flere sammensætninger, der tilsættes til en sekventeringsreaktion til derved ændring af det første påviselige mønster, omfatter tilsætning afen eller flere af en spaltningsforbindelse og et bindingspartner-påvisningsdel-konjugat.
12. Sammensætning til anvendelse i fremgangsmåden ifølge krav 1, som omfatter: a) et første nukleotidkonjugat, der er bundet til en første fluorofor og påviseligt i en første kanal; b) et andet nukleotidkonjugat, der er bundet til en anden fluorofor og påviseligt i en anden kanal; c) et tredje nukleotidkonjugat, der er påviseligt i den første kanal og den anden kanal, hvor det tredje nukleotidkonjugat er en blanding af konjugater, der adskiller sig ved at være bundet til en tredje fluorofor, der er påviselig i den første kanal, eller en fjerde fluorofor, der er påviselig i den anden kanal; og d) et fjerde nukleotidkonjugat, der mangler en fluorescerende del.
13. Sammensætning ifølge krav 12, hvor de fire fluoroforer er forskellige fluorescerende dele.
14. Sammensætning ifølge krav 12, hvor den første fluorofor er identisk med den tredje fluorofor, og den anden fluorofor er identisk med den fjerde fluorofor.
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