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DK3192877T3 - Vesikulær adapter og anvendelser deraf i nukleinsyrebibliotekskonstruktion og -sekvensering - Google Patents

Vesikulær adapter og anvendelser deraf i nukleinsyrebibliotekskonstruktion og -sekvensering Download PDF

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DK3192877T3
DK3192877T3 DK14901593.5T DK14901593T DK3192877T3 DK 3192877 T3 DK3192877 T3 DK 3192877T3 DK 14901593 T DK14901593 T DK 14901593T DK 3192877 T3 DK3192877 T3 DK 3192877T3
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vesicular
strand
region
stranded
terminal
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Yuan Jiang
Jing Guo
Xiaojun Ji
Chunyu Geng
Kai Tian
Xia Zhao
Huaiqian Xu
Wenwei Zhang
Hui Jiang
Radoje Drmanac
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Mgi Tech Co Ltd
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Claims (14)

1. Vesikulær oligonukleotid-adapter til konstruktion af et nukleinsyrebibliotek, der omfatter: et 5’-ende parret dobbeltstrenget område ved en første terminal af adapteren; et 3’-ende parret dobbeltstrenget område ved en anden terminal af adapteren, der omfatter en første streng og en anden strengkomplementaritet med hinanden, hvor den første streng omfatter et overhæng ved 3’-enden deraf og den anden streng omfatter en phosphoryleret base ved 5’-enden deraf for således at tilvejebringe en klæbende terminal; og et vesikulært ikke-parret område mellem det 5’-ende parrede dobbeltstrengede område og det 3’-ende parrede dobbeltstrengede område, hvor det vesikulære ikke-parrede område omfatter en første streng og en anden streng ikke-komplementær med hinanden og den første streng er af en længde, der er længere end længden af den anden streng, hvor den vesikulære adapter er af en længde på mindst 20 nt, fortrinsvis 25 til 50 nt, og mere fortrinsvis 30 til 45 nt.
2. Vesikulær oligonukleotid-adapter ifølge krav 1, hvor den klæbende terminal af det 3’-ende parrede dobbeltstrengede område har en enkelt basehale.
3. Vesikulær oligonukleotid-adapter ifølge krav 2, hvor den enkelte basehale er thymin (T).
4. Vesikulær oligonukleotid-adapter ifølge et hvilket som helst af kravene 1 til 3, hvor den første streng af det vesikulære ikke-parrede område er mindst 5 til 30 nt længere end den anden streng af det vesikulære ikke-parrede område.
5. Vesikulær oligonukleotid-adapter ifølge et hvilket som helst af kravene 1 til 4, hvor det 5’-ende parrede dobbeltstrengede område også har en klæbende terminal.
6. Vesikulær oligonukleotid-adapter ifølge et hvilket som helst af kravene 1 til 5, hvor det 5’-ende parrede dobbeltstrengede område har en klæbende terminal på 1 til 3 ikke-komplementære baser.
7. Vesikulær oligonukleotid-adapter ifølge et hvilket som helst af kravene 1 til 6, der omfatter en sense-streng og en antisense-streng og er af en struktur med formlen I 5’ende terminalen til 3’-ende terminalen: Y0-Y1-Y2 (I) hvor Y0 repræsenterer det 5’-ende parrede dobbeltstrengede område og er af en længde på ΙΟΙ 5 nt, fortrinsvis 11 nt; Y1 repræsenterer et ikke-parret dobbeltstrenget område, hvis sense-streng er af en længde på 5-30 nt længere end antisense-strengens længde; Y2 repræsenterer det 3’ende parrede dobbeltstrengede område.
8. Kit til konstruktion af et nukleinsyrebibliotek, der omfatter: en beholder; en vesikulær oligonukleotid-adapter ifølge et hvilket som helst af kravene 1 til 7 til konstruktion af et bibliotek, hvor den vesikulære oligonukleotid-adapter er indeholdt i beholderen; en første primer med den samme sekvens som mindst en del af den første streng af det vesikulære ikke-parrede område; en anden primer, der specifikt parres med den anden streng af det vesikulære ikke-parrede område og en anvisning.
9. Kit ifølge krav 8, hvor den første primer også er tilvejebragt som en sekvenseringsprimer.
10. Anvendelse af en vesikulær oligonukleotid-adapter ifølge krav 1 til konstruktion af et nukleinsyrebibliotek, hvor et dobbelt-strenget DNA-fragment er ligeret med den vesikulære oligonukleotid-adapter ved hver terminal deraf for således at tilvejebringe en struktur K1-K2-K3, hvori Kl og K3 hver repræsenter en vesikulær adapter ligeret via den klæbende terminal af det 3’-ende parrede dobbeltstrengede område, hvor K2 repræsenterer en vilkår DNA-sekvens (sekvens af et fragment, der skal sekvenseres); hvori henholdsvis Kl og K2 er forbundet med K2 ved to terminaler af K2.
11. Anvendelse af en oligonukleotid-adapter ifølge krav 10, hvor et kit ifølge krav 8 anvendes og den første og den anden af kittets primere anvendes til PCR-amplifikation ved anvendelse af strukturen K1-K2-K3.
12. Fremgangsmåde til konstruktion af et cyklisk enkeltstrenget bibliotek, der omfatter: (a) endereparation af et dobbelt-strenget DNA-fragment for at opnå et dobbelt-strenget DNA-fragment med afstumpede terminaler; (b) tilføjelse af en adenin- (A) base til hver 3’-ende af det dobbelt-strengede DNA-fragment med de afstumpede terminaler opnået i (a), for at opnå et dobbelt-strenget DNA-fragment med en A-base ved hver 3’-ende deraf; (c) ligering af en vesikulær oligonukleotid-adapter ifølge krav 3 til hver terminal af det dobbelt-strengede DNA-fragment med A-basen ved hver 3’-ende deraf opnået i (b) for at opnå et dobbelt-strenget DNA-fragment ligeret med den vesikulære oligonukleotid-adapter ved hver terminal deraf; (d) anvendelse af det dobbelt-strengede DNA-fragment ligeret med den vesikulære oligonukleotid-adapter ved hver terminal deraf opnået i (c), som en template for PCR- amplifikation med et par primere som defineret i krav 8 for således at opnå et DNA-amplificeret produkt, hvor et af primerparrene er mærket med biotin; (e) isolering af det enkelt-strengede DNA mærket med biotin fra det amplificerede dobbeltstrengede DNA-produkt opnået i (d) ved anvendelse af kugler coatet med avidin via ’’avidin-biotin-” kombination, hvorved der opnås det enkelt-strengede DNA minus biotin til cyklisering; (f) udsættelse af det enkelt-strengede DNA minus biotin opnået i (e) for cyklisering i tilstedeværelse af et cykliserende enkelt-strenget molekyle.
13. Fremgangsmåde ifølge krav 12, der endvidere omfatter: (g) digestion af ikke-cykliserede DNA’er opnået i (f) med nukleaser, der specifikt digesterer DNA’er for at opnå et præprodukt; og (h) oprensning af præproduktet opnået i (g) for at opnå det cykliske enkeltstrengede bibliotek.
14. Fremgangsmåde ifølge krav 13, hvor nukleaserne anvendt i (g) omfatter en første exonuklease, der specifikt digesterer lineære enkelt-strengede DNA’er og en anden exonuklease, der specifikt digesterer lineære dobbeltstrengede DNA’er.
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