DK2644707T3 - RNase-H-baserede assays, som anvender modificerede RNA-monomere. - Google Patents
RNase-H-baserede assays, som anvender modificerede RNA-monomere. Download PDFInfo
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- DK2644707T3 DK2644707T3 DK13173388.3T DK13173388T DK2644707T3 DK 2644707 T3 DK2644707 T3 DK 2644707T3 DK 13173388 T DK13173388 T DK 13173388T DK 2644707 T3 DK2644707 T3 DK 2644707T3
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- primer
- rnase
- cleavage
- dna
- enzyme
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Claims (22)
1. Fremgangsmåde til amplificering af en mål-DNA-sekvens, hvor fremgangsmåden omfatter trinnene at: a) tilvejebringe en reaktionsblanding omfattende (i) en oligonukleotidprimer, der har et spaltningsdomæne positioneret 5' af en blokeringsgruppe, hvor blokeringsgruppen er bundet på eller i nærheden af enden af 3'-enden af oligonukleotidprimeren, hvor blokeringsgruppen inhiberer primerforlængelse og/eller pri-meren fra at tjene som en skabelon til DNA-syntese, (ii) en prøvenukleinsyre som måske eller måske ikke har målsekvensen, (iii) et spaltningsenzym, hvor spaltningsenzymet er et termostabilt RNase-H2-enzym, som opnår øget aktivitet ved de forhøjede temperaturer benyttet i reaktionen og har reduceret aktivitet ved lavere temperaturer, og (iv) en polymerase; b) hybridisere primeren til mål-DNA-sekvensen for at danne et dobbeltstrenget substrat; c) spalte den hybridiserede primer med spaltningsenzymet på et sted indenfor eller tilstødende til spaltningsdomænet for at fjerne blokeringsgruppen fra primeren; og d) forlænge primeren med polymerasen.
2. Fremgangsmåde ifølge krav 1, hvor RNase-FI2-enzymet er reversibelt inaktiveret ved kemisk modifikation eller med et blokeringsantistof.
3. Fremgangsmåde ifølge krav 1, hvor blokeringsgruppen er (a) bundet til 3'-terminalnukleotidet af primeren, eller (b) er bundet 5' af 3'-terminalresten.
4. Fremgangsmåde ifølge krav 3(b), hvor blokeringsgruppen indbefatter én eller flere abasiske rester eller modificerede nukleosider, fortrinsvis er den abasiske rest en C3-spacer og det modificerede nukleosid er en 2'-0-methyl-riboserest.
5. Fremgangsmåde ifølge krav 1, hvor blokeringsgruppen indbefatter et mærke, som tillader detektion af amplifikationsreaktionen, fortrinsvis er mærket en fluorofor, et slukningsmiddel (ENG: quencher), biotin, en hapten eller en masse-markør til detektion af amplifikationsreaktionen ved massespektrometri.
6. Fremgangsmåde ifølge krav 1, hvor spaltningsdomænet er en kontinuerlig sekvens af 3 eller flere RNA-rester, fortrinsvis omfatter spaltningsdomænet yderligere én eller flere af de følgende dele: en DNA-rest, en abasisk rest, et modificeret nukleosid, eller en modificeret phosphat-internukleotidbinding.
7. Fremgangsmåde ifølge krav 2, hvor spaltningsdomænet er én enkelt RNA-rest eller to tilstødende RNA-rester.
8. Fremgangsmåde ifølge krav 2, hvor spaltningsdomænet mangler en RNA-rest, fortrinsvis omfatter spaltningsdomænet ét eller flere 2'-modificerede nukleosider.
9. Fremgangsmåde ifølge krav 8, hvor det 2'-modificerede nukleosid er ét enkelt 2'-fluornukleosid, eller, hvor spaltningsdomænet er to tilstødende 2'-fluornukleosidrester.
10. Fremgangsmåde ifølge krav 8, hvor spaltningsreaktionen udføres under tilstede- værelse af én eller flere af de følgende divalente kationer: mangan, cobalt, nikkel eller zink, eventuelt, er magnesium også tilstede i reaktionsblandingen.
11. Fremgangsmåde ifølge krav 7, hvor en sekvens indenfor eller flankerende spaltningsdomænet, indeholder én eller flere internukleosidbindinger resistente over for nuklea-sespaltning, fortrinsvis er den nukleaseresistente binding phosphorthioat, phosphor-dithioat, methylphosphonat eller en abasisk rest og er eventuelt på 3'-siden af spaltningsdomænet.
12. Fremgangsmåde ifølge krav 1, yderligere omfattende en anden primer i modsat orientering af den første primer til at støtte PCR, fortrinsvis er den anden primer en ikke-modificeret DNA-primer og eventuelt omfattende et spaltningsdomæne positioneret 5' af en blokeringsgruppe, blokeringsgruppen er bundet på eller i nærheden af enden af 3'-enden af oligonukleotidprimeren, hvor blokeringsgruppen forhindrer primerforlængelse.
13. Fremgangsmåde ifølge krav 12, hvor PCR-assayet anvendes til at: (a) skelne mellem variant-alleler; eller (b) kvantificere hyppigheden af mål-nukleinsekvensen i prøven.
14. Fremgangsmåde ifølge krav 13, hvor (a) et sekundært mutationssted er inkorporeret indenfor eller flankerende spaltningsdomænet for at forøge detektion af variant-allelen, eller (b) et modificeret nukleosid er inkorporeret indenfor eller flankerende spaltningsdomænet for at forøge detektion af variant-allelen, fortrinsvis er det modificerede nukleosid en 2'-0-methyl-riboserest.
15. Fremgangsmåde ifølge krav 13, hvor en nukleaseresistent binding er inkorporeret på 3'-siden af spaltningsdomænet.
16. Fremgangsmåde ifølge krav 12, hvor PCR-assayet er et primer-probe-PCR-assay.
17. Fremgangsmåde ifølge krav 16, hvor primeren, der har et 5'-mærkedomæne, indbefatter et spaltningsdomæne, fortrinsvis et RNase-H-spaltningsdomæne, mere fortrinsvis et RNase-H2-spaltningsdomæne.
18. Fremgangsmåde til amplificering af mål-DNA-sekvens, hvor fremgangsmåden omfatter trinnene at: a) tilvejebringe en reaktionsblanding omfattende: (i) en oligonukleotidprimer, der har et spaltningsdomæne positioneret 5' af en blokeringsgruppe, blokeringsgruppen er bundet opstrøms for 5' af 3'-terminalresten af oligonukleotidprimeren, hvor blokeringsgruppen inhiberer primerforlængelse og/eller primeren fra at tjene som en skabelon til DNA-syntese, (ii) en prøvenukleinsyre som måske eller måske ikke har målsekvensen, (iii) et termostabilt RNase-H2-enzym som opnår øget aktivitet ved de forhøjede temperaturer i reaktionen og har reduceret aktivitet ved lavere temperaturer og (iv) en polymerase; b) hybridisere primeren til mål-DNA-sekvensen for at danne et dobbeltstrenget substrat; c) spalte den hybridiserede primer med det termostabile RNase-H2-enzym på et sted indenfor eller tilstødende til spaltningsdomænet for at fjerne blokeringsgruppen fra prime-ren; og d) forlænge primeren med polymerasen.
19. Fremgangsmåde ifølge krav 7 eller 18, hvor spaltningsdomænet er én enkelt RNA-rest og blokeringsgruppen omfatter to eller flere abasiske rester.
20. Fremgangsmåde ifølge krav 18 eller 19, hvor en region 3' til RNA-resten af primeren omfatter en sekvens Dl-x-x-D2, hvor Dl og D2 er DNA-baser og x er en abasisk rest.
21. Anvendelse af en primer til DNA-replikation, der har en 5'-ende og en 3'-ende, primeren omfattende: a) en ikke-aktiveret konfiguration når primeren er ikke-hybridiseret til en DNA-sekvens af interesse, den ikke-aktiverede konfiguration omfattende: (i) et RNase-FI2-spaltningsdomæne omfattende én eller flere 2'-modificerede nukleo-sidrester, og en blokeringsgruppe bundet på eller i nærheden af 3'-enden af primeren; eller (ii) et RNase-FI2-spaltningsdomæne, og en blokeringsgruppe bundet på eller i nærheden af 3'-enden af primeren, som inkorporerer et mærke; eller (iii) et RNase-FI2-spaltningsdomæne, og en blokeringsgruppe placeret 5' til 3'-terminalnukleotidresten af primeren; og b) en aktiveret konfiguration når primeren er hybridiseret til DNA-sekvensen af interesse, hvor den aktiverede konfiguration er fri af blokeringsgruppen og er i stand til at støtte DNA-replikation; i fremgangsmåden ifølge et hvilket som helst af krav 1-20.
22. Anvendelse ifølge krav 21, hvor den 2'-modificerede nukleosidrest af (i) er en 2'-fluornukleosid.
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