DK2665833T3 - Arbejdsprocedure for påvisning af ligander ved anvendelse af nukleinsyrer - Google Patents
Arbejdsprocedure for påvisning af ligander ved anvendelse af nukleinsyrer Download PDFInfo
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- DK2665833T3 DK2665833T3 DK12702099.8T DK12702099T DK2665833T3 DK 2665833 T3 DK2665833 T3 DK 2665833T3 DK 12702099 T DK12702099 T DK 12702099T DK 2665833 T3 DK2665833 T3 DK 2665833T3
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- DK
- Denmark
- Prior art keywords
- ligase
- oligonucleotide
- seq
- ligation
- probe
- Prior art date
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
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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
- C12Q1/6804—Nucleic acid analysis using immunogens
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
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- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Genetics & Genomics (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Claims (14)
1. Fremgangsmåde til ligering af mindst to oligonukleotider for at fremstille et ligeret oligonukleotid og amplifikation af det ligerede oligonukleotid, hvor ligering og amplifikation sker i en enkelt reaktionsblanding, hvilken fremgangsmåde omfatter følgende trin i kombination: a) etablering af kontakt mellem et målprotein eller en målanalyt og mindst en første og en anden probe, hvor hver probe har bindingsspecificitet for proteinet eller analytten og grænser op til mindst én type oligonukleotid; b) ligering af oligonukleotiderne på den første og den anden probe til hinanden ved anvendelse af (i) en ligase valgt fra gruppen bestående af ligasen ifølge SEQ ID NO: 77, ligasen ifølge SEQ ID NO: 78, ligasen ifølge SEQ ID NO: 79, ligasen ifølge SEQ ID NO: 80, ligasen ifølge SEQ ID NO: 81, ligasen ifølge SEQ ID NO: 82 og kombinationer deraf, og (ii) et splint-oligonukleotid, hvor splint-oligonukleotidet omfatter en 3'-ende med en længde på fire til ni baser og en 5'-ende med en længde på fire til ni baser, for at fremstille en målnukleinsyre og amplificere målnukleinsyren; og c) påvisning af den amplificerede målnukleinsyre.
2. Fremgangsmåde ifølge krav 1, hvor mindst ét af oligonukleotiderne omfatter mindst tre nukleotider.
3. Fremgangsmåde ifølge krav 1, hvor oligonukleotiderne på den første og den anden probe er mindst delvist komplementære til hinanden.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1-3, hvor det ligerede oligonukleotid amplificeres ved anvendelse af polymerasekædereaktionen (PCR), og hvor det amplificerede ligerede oligonukleotid påvises ved anvendelse af kvantitativ PCR (qPCR).
5. Fremgangsmåde til påvisning af et målstof i en prøve, hvor: a) binding af en første og en anden probe, som hver binder sig specifikt til målstoffet, hvor hver af proberne omfatter en oligonukleotidhale; b) ligering af den første og den anden oligonukleotidhale ved anvendelse af (i) en ligase valgt fra gruppen bestående af ligasen ifølge SEQ ID NO: 77, ligasen ifølge SEQ ID NO: 78, ligasen ifølge SEQ ID NO: 79, ligasen ifølge SEQ ID NO: 80, ligasen ifølge SEQ ID NO: 81, ligasen ifølge SEQ ID NO: 82 og kombinationer deraf, og (ii) et splint-oligonukleotid, hvor splint-oligonukleotidet omfatter en 3'-ende med en længde på fire til ni baser og en 5'-ende med en længde på fire til ni baser, idet der derved fremstilles en ligeret oligonukleotidtemplate; og c) udførelse af en polymerasekædereaktion (PCR) med oligonukleotidtemplaten over det første og det andet oligonukleotid for at kvantificere templaten, hvor trin b) og c) udføres i samme reaktionsblanding.
6. Fremgangsmåde ifølge krav 5, hvor den første og den anden oligonukleotidhale har en længde på mindst 3 nukleotider.
7. Fremgangsmåde ifølge krav 6, hvor 3'-enden af splint-oligonukleotidet overlapper med mindst 3 nukleotider fra den første oligonukleotidhale, og/eller 5'-enden af splint-oligonukleotidet overlapper med mindst 3 nukleotider fra den anden oligonukleotidhale.
8. Fremgangsmåde ifølge krav 6 eller 7, hvor 3'- og 5'-enderne af splint-oligonukleotidet er symmetriske eller asymmetriske i forhold til hinanden.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 1 eller 5-8, hvor den første og/eller den anden probe endvidere omfatter et antistof eller en antistofdel, som er specifik(t) for målstoffet.
10. Fremgangsmåde ifølge et hvilket som helst af kravene 1-9, hvor splint-oligonukleotidet er blokeret i sin 3'-ende.
11. Fremgangsmåde ifølge et hvilket som helst af kravene 1-10, hvor ATP udelades fra reaktionsblandingen i trin b), og hvor der etableres kontakt mellem ligasen og adenosintriphosphat inden anvendelse i trin b).
12. Fremgangsmåde ifølge et hvilket som helst af kravene 8-11, hvor ligasen inaktiveres efter ligering ved anvendelse af varme.
13. Fremgangsmåde ifølge et hvilket som helst af kravene 8-12, hvor templaten kvantificeres ved anvendelse af realtids-PCR.
14. Fremgangsmåde ifølge et hvilket som helst af kravene 8-13, hvor realtids-PCR-assayet er et TaqMan-assay.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161433475P | 2011-01-17 | 2011-01-17 | |
| PCT/US2012/021585 WO2012099896A2 (en) | 2011-01-17 | 2012-01-17 | Workflow for detection of ligands using nucleic acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2665833T3 true DK2665833T3 (da) | 2017-07-24 |
Family
ID=45561104
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK17166444.4T DK3216878T3 (da) | 2011-01-17 | 2012-01-17 | Arbejdsprocedure til påvisning af ligander ved anvendelse af nukleinsyrer |
| DK12702099.8T DK2665833T3 (da) | 2011-01-17 | 2012-01-17 | Arbejdsprocedure for påvisning af ligander ved anvendelse af nukleinsyrer |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK17166444.4T DK3216878T3 (da) | 2011-01-17 | 2012-01-17 | Arbejdsprocedure til påvisning af ligander ved anvendelse af nukleinsyrer |
Country Status (5)
| Country | Link |
|---|---|
| US (4) | US20120196294A1 (da) |
| EP (3) | EP3216878B1 (da) |
| DK (2) | DK3216878T3 (da) |
| ES (2) | ES2632768T3 (da) |
| WO (1) | WO2012099896A2 (da) |
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- 2012-01-17 US US13/352,237 patent/US20120196294A1/en not_active Abandoned
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- 2012-01-17 ES ES17166444T patent/ES2728743T3/es active Active
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| DK3216878T3 (da) | 2019-06-11 |
| ES2632768T3 (es) | 2017-09-15 |
| EP3216878B1 (en) | 2019-04-03 |
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| US20200048695A1 (en) | 2020-02-13 |
| EP3567121B1 (en) | 2023-08-30 |
| US11072824B2 (en) | 2021-07-27 |
| EP2665833A2 (en) | 2013-11-27 |
| US20210324460A1 (en) | 2021-10-21 |
| US20160153035A1 (en) | 2016-06-02 |
| ES2728743T3 (es) | 2019-10-28 |
| WO2012099896A3 (en) | 2012-09-20 |
| WO2012099896A2 (en) | 2012-07-26 |
| EP3567121A1 (en) | 2019-11-13 |
| US20120196294A1 (en) | 2012-08-02 |
| US10472671B2 (en) | 2019-11-12 |
| EP2665833B1 (en) | 2017-04-19 |
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