DK2828408T3 - Fremgangsmåde til registrering af enkeltnukleotider - Google Patents
Fremgangsmåde til registrering af enkeltnukleotider Download PDFInfo
- Publication number
- DK2828408T3 DK2828408T3 DK14718147.3T DK14718147T DK2828408T3 DK 2828408 T3 DK2828408 T3 DK 2828408T3 DK 14718147 T DK14718147 T DK 14718147T DK 2828408 T3 DK2828408 T3 DK 2828408T3
- Authority
- DK
- Denmark
- Prior art keywords
- stranded
- nucleotide
- oligonucleotide
- capture system
- region
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
- B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
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- Clinical Laboratory Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Claims (20)
1. Fremgangsmåde til bestemmelse af sekvensen af nukleotidbaser i en polynu-kleotidanalyt, kendetegnet ved trinene: (1) generering af en strøm af enkelte nukleotidbasetrifosfater fra analytten ved progressiv pyrofosforolyse under tilstedeværelse af et pyrofosforolyseenzym; (2) frembringelse af opfangede molekyler ved reagering af hvert enkelt nukleotidbasetrifosfat under tilstedeværelse af en polymerase og en ligase med et opfangningssystem bestående af enten (i) to komponenter omfattende (a) et første nukleotid omfattende et dobbeltstrenget område og et enkeltstrenget område og (b) et andet enkeltstrenget oligonu-kleotid, hvis nukleotidbasesekvens er i det mindste delvist komplementært til det første oligonukleotids enkeltstrengede område, eller (ii) et enkelt oligonukleotid omfattende et enkeltstrenget nukleotidområde, hvis ender er fæstnet til to forskellige dobbeltstrengede oligonukleotidområder, og hvori opfangningssystemet er mærket med detekterbare elementer i en ikke-detekterbar tilstand; (3) frigørelse af de detekterbare elementer fra hvert opfanget molekyle i en detekterbar tilstand under anvendelse af en exonuklease eller en polymerases exonukleaseak-tivitet; og (4) registrering af de således frigjorte detekterbare elementer og bestemmelse af sekvensen af nukleotidbaser deraf.
2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at polynukleotidanalytten er bundet til en overflade.
3. Fremgangsmåde ifølge krav 1 eller 2, kendetegnet ved, at pyrofosforolyseen-zymet hverken udviser exonuklease- eller endonukleaseadfærd under reaktionsbetingelserne i trin (1).
4. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at trin (1) udføres under ikke-ekvilibriumtilstande under tilstedeværelse af et strømmende vandigt medium omfattende pyrofosforolyseenzymet, pyrofosfata-nionen og magnesiumkationer, og hvori de enkelte nukleotidbasetrifosfater kontinuerligt fjernes fra reaktionszonen, hvor de genereres.
5. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at mellem trinene (1) og (2) ødelægges alle resterende pyrofosfatanioner ved hjælp af en pyrofosfatase.
6. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at i opfangningssystemet (i) er det første oligonukleotid j-formet.
7. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at i opfangningssystemet (i) er den samlede længde af den første oligonukleotid fra 20 til 100 nukleotidbaser.
8. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at i opfangningssystemet (i) er de detekterbare elementer fæstnet til det andet nu-kleotid og er fluoroforer, som er blevet quenchet med mindst en quencher.
9. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at opfangningssystemet (i) anvendes og omfatter fire forskellige første oligonu-kleotidtyper, der har enkeltstrengede områder af fire forskellige sekvenser, og hvori nukleotidbasen tilstødende det dobbeltstrengede område på det enkeltstrengede område i hver første oligonukleotidtype er en forskellig af de fire nu-kleotidbasetyper med karakteristisk DNA eller RNA.
10. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at opfangningssystemet (i) anvendes og omfatter fire forskellige andre oligonu-kleotidtyper, hver med en sekvens, som er komplementær til en del af en af de fire forskellige enkeltstrengede områder i de fire forskellige første oligonukleotider og hver mærket med et forskelligt detekterbart element.
11. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at opfangningssystemet (i) anvendes, og hver anden oligonukleotidtype mærkes med en forskellig fluorofor, der fluorescerer ved en forskellig bølgelængde.
12. Fremgangsmåde ifølge ethvert af krav 1 til 5, kendetegnet ved, at i opfangningssystemet (ii) består hvert dobbeltstrenget oligonukleotidområde af 10 til 30 nukleotidpar.
13. Fremgangsmåde ifølge ethvert af krav 1 til 5 og 12, kendetegnet ved, at i opfangningssystemet (ii) er op til 10 nukleotidpar i et dobbeltstrenget oligonukleotidområde mærket med en fluorofor.
14. Fremgangsmåde ifølge ethvert af krav 1 til 5, 12 og 13, kendetegnet ved, at i opfangningssystemet (ii) er op til 10 nukleotidpar i et dobbeltstrenget oligo-nukleotidområde mærket med en quencher.
15. Fremgangsmåde ifølge ethvert af krav 1 til 5 og 12 til 14, kendetegnet ved, at i opfangningssystemet (ii) anvendes to diskrete dobbeltstrengede oligonu-kleotidområder, som hver omfatter ender fjernt fra det enkeltstrengede nukleoti-dområde, hvilke ender danner lukket sløjfe.
16. Fremgangsmåde ifølge ethvert af krav 1 til 5 og 12 til 15, kendetegnet ved, at i opfangningssystemet (ii) kan de dobbeltstrengede oligonukleotidområder udledes af en enkeltstrenget oligonukleotidforløber ved foldning af ender tilbage på dem selv for at efterlade et mellemrum omfattende det enkeltstrengede nukleoti-dområde.
17. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at opfangningssystemet omfatter mindst et restriktionsenzymgenkendelsessted.
18. Fremgangsmåde ifølge krav 17, kendetegnet ved, at restriktionsenzymgenkendelsesstedet skabes ved at knytte det enkelte nukleotidtrifosfat til opfangningssystemet.
19. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at trin (4) omfatter registrering af fluorescens udsendt af fluoroforerne.
20. Fremgangsmåde ifølge ethvert af de foregående krav, kendetegnet ved, at mindst et af trinene (1) til (4) udføres i en strøm af mikroskopiske dråber.
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| GB201306444A GB201306444D0 (en) | 2013-04-09 | 2013-04-09 | Single nucleotide detection method |
| PCT/GB2014/051105 WO2014167323A1 (en) | 2013-04-09 | 2014-04-09 | Single nucleotide detection method |
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| DK2828408T3 true DK2828408T3 (da) | 2015-12-14 |
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| GB (1) | GB201306444D0 (da) |
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| GB201310584D0 (en) * | 2013-06-13 | 2013-07-31 | Base4 Innovation Ltd | Droplet storage 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 |
| GB201501012D0 (en) * | 2015-01-21 | 2015-03-04 | Base4 Innovation Ltd | Improved droplet sequencing apparatus and method |
| EP3115109A1 (en) | 2015-07-06 | 2017-01-11 | Base4 Innovation Limited | Droplet sequencing device |
| EP3207982A1 (en) | 2016-02-17 | 2017-08-23 | Base4 Innovation Limited | Improved droplet sequencing apparatus and method |
| EP3211092B1 (en) | 2016-02-24 | 2019-03-27 | Base4 Innovation Ltd | Single nucleotide detection method |
| EP3269444A1 (en) | 2016-07-14 | 2018-01-17 | Base4 Innovation Ltd | Method of identifying droplets in a stack and an associated sequencer |
| JP2019532623A (ja) | 2016-09-02 | 2019-11-14 | ベース４ イノベーション リミテッド | 単一ヌクレオチド検出方法および関連プローブ |
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| EP2828408A1 (en) | 2015-01-28 |
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