DK2796552T3 - Fremgangsmåder og apparater til syntetisering nukleinsyrer - Google Patents

Fremgangsmåder og apparater til syntetisering nukleinsyrer Download PDF

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DK2796552T3
DK2796552T3 DK14169758.1T DK14169758T DK2796552T3 DK 2796552 T3 DK2796552 T3 DK 2796552T3 DK 14169758 T DK14169758 T DK 14169758T DK 2796552 T3 DK2796552 T3 DK 2796552T3
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nucleotide
leu
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William J Efcavitch
Suhaib Siddiqi
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Molecular Assemblies Inc
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07019—Polynucleotide adenylyltransferase (2.7.7.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (12)

1. Fremgangsmåde til syntetisering af et oligonukleotid, hvilken fremgangsmåde omfatter: eksponering af en nukleinsyre bundet til en fast bærer til en nukleotid-analog i nærvær af et nukleotidyl-transferase-enzym, og i fravær af en nukleinsyre-template, hvor nukleotidanalogen omfatter en umodificeret 3' hydroxyl, og en spaltelig afsluttende gruppe omfattende en aminosyre, hvori den spaltelige afsluttende gruppe blokkerer nukleotidyl-transferase-aktivitet, men ved spaltning resulterer i et nukleotidsubstrat for nukleotidyl-transferase.
2. Fremgangsmåden ifølge krav 1, hvor nukleotidanalogen omfatter en ribose-sukker eller en deoxyribose-sukker.
3. Fremgangsmåden ifølge krav 1 eller krav 2, hvor nukleotidsubstratet omfatter en base valgt fra gruppen bestående af adenin, guanin, cytosin, thymin, og uracil.
4. Fremgangsmåden ifølge et hvilket som helst af kravene 1 til 3, hvor nukleotidyl-transferasen: a) omfatter en proteinsekvens, der er mindst ca. 90% identisk med SEQ ID NO. 1, SEQ ID NO. 3, eller SEQ ID NO: 5, eller b) stammer fra en organisme med en nukleotidsekvens, der er mindst 90% identisk med SEQ ID NO. 2, SEQ ID NO. 4, eller SEQ ID NO. 6.
5. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor den spaltelige afsluttende gruppe: a) inhiberer inkorporeringen afen anden nukleotidanalog; b) omfatter en ladet del; c) omfatter en negativt ladet del; d) er kemisk spaltelig, fotolytisk spaltelig, elektrokemisk spaltelig, eller biologisk spaltelig; og / eller e) omfatter en del, der danner et cyklisk bi-produkt ved spaltning fra nukleotidanalogen.
6. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor nukleotidanalogen: a) har strukturen:
, eller
, eller
, eller
, eller
hvor n = 2 eller 3, og -X- er -O-, -S-, -NH- eller -CH2-; eller b) har strukturen:
, eller
, eller
, eller
, eller
hvor n = 2 eller 3, og -X- er -O-, -S-, -NH- eller-CH2-; eller c) har strukturen:
,eller
, eller
, eller
, eller
, eller d) har strukturen:
, eller
, eller
, eller
, eller
7. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor: a) nukleinsyren bundet til den faste bærer eksponeres for nukleotid-analogen i nærvær af en vandig opløsning med et pH på mellem ca. 6,5 og ca. 8,5; b) nukleinsyren bundet til den faste bærer eksponeres for nukleotid-analogen i nærvær af en vandig opløsning ved en temperatur på mellem ca. 35 °C og ca. 39 °C; og / eller c) den faste bærer er en perle, en brønd eller en pind.
8. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, hvor nukleinsyren er enkeltstrenget.
9. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, yderligere omfattende: spaltning af den spaltelige afsluttende gruppe for at frembringe et nativt nukleotid; og udsætning af det native nukleotid for en anden nukleotid-analog i nærvær af et transferase-enzym, og i fravær af en nukleinsyre-template, hvor den anden nukleotidanalog omfatter en 3' hydroxyl på en sukkerring, og en spaltelig afsluttende gruppe.
10. Fremgangsmåden ifølge et hvilket som helst af de foregående krav, yderligere omfattende tilvejebringelse afen vandig opløsning omfattende nukleotidanalogen og nukleotidyl-transferase-enzymet.
11. Et system til syntetisering af et oligonukleotid, hvilket system omfatter: en fast bærer til hvilken en nukleinsyre er bundet; et nukleotidyl-transferase-enzym; et første nukleotid, der omfatter en fri 3' hydroxyl, og en blokerende gruppe, der omfatter en aminosyre, der er bundet til nukleotidet via en spaltelig linker, hvor et andet nukleotid ikke kan kobles af nukleotidyl-transferasen til det første nukleotid via 3' positionen af det første nukleotid, før den blokerende gruppe er fjernet via den spaltelige linker, og hvor systemet ikke indeholder en nukleinsyre-template.
12. Systemet ifølge krav 11, hvor det første nukleotid og det andet nukleotid uafhængigt omfatter en base valgt fra gruppen bestående af adenin, guanin, cytosin, thymin, og uracil.
DK14169758.1T 2013-04-02 2014-05-23 Fremgangsmåder og apparater til syntetisering nukleinsyrer DK2796552T3 (da)

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US201361807327P 2013-04-02 2013-04-02
US201361891162P 2013-10-15 2013-10-15
US14/056,687 US8808989B1 (en) 2013-04-02 2013-10-17 Methods and apparatus for synthesizing nucleic acids

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AU (1) AU2014247928B2 (da)
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