HRP20200523T1 - Sastavljanje dnk posredovano nukleazom - Google Patents
Sastavljanje dnk posredovano nukleazom Download PDFInfo
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- HRP20200523T1 HRP20200523T1 HRP20200523TT HRP20200523T HRP20200523T1 HR P20200523 T1 HRP20200523 T1 HR P20200523T1 HR P20200523T T HRP20200523T T HR P20200523TT HR P20200523 T HRP20200523 T HR P20200523T HR P20200523 T1 HRP20200523 T1 HR P20200523T1
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- Croatia
- Prior art keywords
- nucleic acid
- digested
- complementary
- sequence
- oligonucleotide
- Prior art date
Links
- 101710163270 Nuclease Proteins 0.000 title claims 10
- 230000001404 mediated effect Effects 0.000 title 1
- 108020004707 nucleic acids Proteins 0.000 claims 62
- 102000039446 nucleic acids Human genes 0.000 claims 62
- 150000007523 nucleic acids Chemical class 0.000 claims 62
- 230000000295 complement effect Effects 0.000 claims 30
- 108091034117 Oligonucleotide Proteins 0.000 claims 21
- 238000000034 method Methods 0.000 claims 15
- 108020004414 DNA Proteins 0.000 claims 8
- 239000003795 chemical substances by application Substances 0.000 claims 8
- 108060002716 Exonuclease Proteins 0.000 claims 6
- 102000013165 exonuclease Human genes 0.000 claims 6
- 108020005004 Guide RNA Proteins 0.000 claims 4
- 238000010459 TALEN Methods 0.000 claims 3
- 102000053602 DNA Human genes 0.000 claims 2
- 241000283984 Rodentia Species 0.000 claims 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 claims 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 claims 2
- 238000000338 in vitro Methods 0.000 claims 2
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 claims 2
- 102000004169 proteins and genes Human genes 0.000 claims 2
- 108091008146 restriction endonucleases Proteins 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 1
- 239000012636 effector Substances 0.000 claims 1
- 125000006850 spacer group Chemical group 0.000 claims 1
- 108091006106 transcriptional activators Proteins 0.000 claims 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
Claims (15)
1. In vitro Postupak bešavnog sastavljanja dviju ili više dvolančanih nukleinskih kiselina, naznačen time, što uključuje:
(a) dovođenje u kontakt prve nukleinske kiseline sa najmanje jednim nukleaznim sredstvom da bi se dobila prva digestirana nukleinska kiselina kojoj je uklonjen završni dio;
(b) dovođenje u kontakt prve digestirane nukleinske kiseline sa drugom nukleinskom kiselinom, dvolančanim spojnim oligonukleotidom, i egzonukleazom, pri čemu je spojni oligonukleotid predstavljen linearnom dvolančanom DNK od oko 50 bp do oko 400 bp i uključuje:
(i) prvu komplementarnu sekvencu koja je komplementarna sa prvom digestiranom nukleinskom kiselinom;
(ii) razdjelnik; i
(iii) drugu komplementarnu sekvencu koja je komplementarna sa drugom nukleinskom kiselinom
gdje razdjelnik uključuje sekvencu identičnu sa završnim dijelom,
pri čemu između prve komplementarne sekvence i sekvence identične sa završnim dijelom nema nukleinsko-kiselinskih baza, i između druge komplementarne sekvence i sekvence identične sa završnim dijelom nema nukleinsko-kiselinskih baza, i
pri čemu egzonukleaza izlaže prvu i drugu komplementarnu sekvencu; i
(c) sastavljanje spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i drugom nukleinskom kiselinom, pri čemu se sastavljanjem rekonstituiše završni dio.
2. Postupak iz patentnog zahtjeva 1, naznačen time, što sastavljanje u koraku (c) uključuje:
(i) sparivanje prve komplementarne sekvence spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i druge komplementarne sekvence spojnog oligonukleotida sa drugom nukleinskom kiselinom; i
(ii) povezivanje spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i drugom nukleinskom kiselinom.
3. Postupak iz patentnog zahtjeva 1 ili 2, naznačen time, što korak (a) uključuje još i:
(i) dovođenje u kontakt druge nukleinske kiseline sa drugim nukleaznim sredstvom i egzonukleazom, pri čemu drugo nukleazno sredstvo siječe drugu nukleinsku kiselinu na drugom ciljnom mjestu da se proizvede druga digestirana nukleinska kiselina koja sadrži nukleotidnu sekvencu komplementarnu sa drugom komplementarnom sekvencom spojnog oligonukleotida, pri čemu se prva digestirana nukleinska kiselina sastavlja sa drugom digestiranom nukleinskom kiselinom; ili
(ii) dovođenje u kontakt druge nukleinske kiseline sa restrikcionim enzimom i egzonukleazom, pri čemu restrikcioni enzim siječe drugu nukleinsku kiselinu da bi se proizvela druga digestirana nukleinska kiselina koja uključuje nukleotidnu sekvencu komplementarnu sa drugom komplementarnom sekvencom u spojnom oligonukleotidu, pri čemu se prva digestirana nukleinska kiselina sastavlja sa drugom digestiranom nukleinskom kiselinom.
4. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što korak (b) uključuje još i produžavanje 3’ kraja prve digestirane nukleinske kiseline.
5. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što se spojni oligonukleotid sastavlja sa prvom digestiranom nukleinskom kiselinom i drugom nukleinskom kiselinom u istoj reakciji ili sekvencijalno.
6. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što najmanje jedno nukleazno sredstvo uključuje Cas protein i vodičku RNK (gRNK) (gRNK-Cas ompleks), zinc finger nukleazu, ili efektorsku nukleazu sličnu transkripcijskom aktivatoru (TALEN).
7. In vitro postupak bešavnog sastavljanja dviju ili više dvolančanih nukleinskih kiselina, naznačen time, što uključuje:
(a) dovođenje u kontakt prve nukleinske kiseline sa prvim nukleaznim sredstvom da bi nastala prva digestirana nukleinska kiselina sa uklonjenim završnim dijelom;
(b) dovođenje u kontakt druge nukleinske kiseline sa drugim nukleaznim sredstvom da bi nastala druga digestirana nukleinska kiselina;
(c) dovođenje u kontakt prve digestirane nukleinske kiseline i druge digestirane nukleinske kiseline sa dvolančanim spojnim oligonukleotidom i egzonukleazom, pri čemu je spojni oligonukleotid predstavljen linearnom dvolančanom DNK dugom od oko 50 bp do oko 400 bp i uključuje:
(i) prvu komplementarnu sekvencu koja je komplementarna sa prvom digestiranom nukleinskom kiselinim;
(ii) razdjelnik; i
(iii) drugu komplementarnu sekvencu koja je komplementarna sa drugom digestiranom nukleinskom kiselinom,
pri čemu razdjelnik uključuje sekvencu koja je identična sa završnim dijelom,
pri čemu između prve komplementarne sekvence i sekvence koja je identična sa završnim dijelom nema nukleinsko-kiselinskih baza, i između druge komplementarne sekvence i sekvence koja je identična sa završnim dijelom nema nukleinsko-kiselinskih baza, i
pri čemu egzonukleaza izlaže prvu i drugu komplementarnu sekvencu; i
(d) sastavljanje spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i drugom digestiranom nukleinskom kiselinom, pri čemu se sastavljanjem rekonstituiše završni dio.
8. Postupak iz patentnog zahtjeva 7, naznačen time, što sastavljanje u koraku (d) uključuje:
(i) sparivanje prve komplementarne sekvence spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i druge komplementarne sekvence spojnog oligonukleotida sa drugom digestiranom nukleinskom kiselinom; i
(ii) povezivanje spojnog oligonukleotida sa prvom digestiranom nukleinskom kiselinom i drugom digestiranom nukleinskom kiselinom.
9. Postupak iz patentnog zahtjeva 7 ili 8, naznačen time, što korak (c) uključuje još i produžavanje 3’ kraja prve digestirane nukleinske kiseline i/ili druge digestirane nukleinske kiseline.
10. Postupak iz bilo kojeg od patentnih zahtjeva 7-9, naznačen time, što se spojni oligonukleotid sastavlja sa prvom digestiranom nukleinskom kiselinom i drugom digestiranom nukleinskom kiselinom u istoj reakciji ili sekvencijalno.
11. Postupak iz bilo kojeg od patentnih zahtjeva 7-10, naznačen time, što prvo nukleazno sredstvo i/ili drugo nukleazno sredstvo uključuje Cas protein i vodičku RNK (gRNK) (gRNK-Cas kompleks), zinc finger nukleazu, ili efektorsku nukleazu sličnu transkripcijskom aktivatoru (TALEN).
12. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što:
(I) prva komplementarna sekvenca spojnog oligonukleotida ima između 15 i 120 komplementarnih baza i druga komplementarna sekvenca spojnog oligonukleotida ima između 15 i 120 komplementarnih baza; i/ili
(II) završni dio ima najmanje 20 bp; i/ili
(III) razdjelnik ima od oko 20 bp do oko 120 bp.
13. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što:
(I) prva nukleinska kiselina, druga nukleinska kiselina, ili obje nukleinske kiseline uključuju bakterijski umjetni kromosom; i/ili
(II) prva nukleinska kiselina, druga nukleinska kiselina, ili obje nukleinske kiseline uključuju humanu DNK, glodavačku DNK, sintetsku DNK, ili njihovu kombinaciju; i/ili
(III) prva nukleinska kiselina, druga nukleinska kiselina, ili obje nukleinske kiseline imaju najmanje10 kb; i/ili
(IV) prva nukleinska kiselina, druga nukleinska kiselina, ili obje nukleinske kiseline imaju najmanje 10 kb, uključuju bakterijski umjetni kromosom, i uključuju humanu DNK, glodavačku DNK, sintetsku DNK, ili njihovu kombinaciju.
14. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što se sastavljaju najmanje tri nukleinske kiseline.
15. Postupak iz bilo kojeg od prethodnih patentnih zahtjeva, naznačen time, što:
(I) spojni oligonukleotid ima od oko 100 bp do oko 300 bp; i/ili
(II) prva komplementarna sekvenca spojnog oligonukleotida ima između 20 i 80 komplementarnih baza i druga komplementarna sekvenca spojnog oligonukleotida ima između 20 i 80 komplementarnih baza.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201462015809P | 2014-06-23 | 2014-06-23 | |
| US201462016400P | 2014-06-24 | 2014-06-24 | |
| US201462036983P | 2014-08-13 | 2014-08-13 | |
| EP18162656.5A EP3354732B1 (en) | 2014-06-23 | 2015-06-23 | Nuclease-mediated dna assembly |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HRP20200523T1 true HRP20200523T1 (hr) | 2020-08-07 |
Family
ID=53525285
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HRP20200523TT HRP20200523T1 (hr) | 2014-06-23 | 2020-03-30 | Sastavljanje dnk posredovano nukleazom |
Country Status (25)
| Country | Link |
|---|---|
| US (5) | US9738897B2 (hr) |
| EP (3) | EP3708663A1 (hr) |
| JP (4) | JP6336140B2 (hr) |
| KR (2) | KR102237151B1 (hr) |
| CN (2) | CN112029787A (hr) |
| AU (3) | AU2015280120B2 (hr) |
| BR (1) | BR112016030145A8 (hr) |
| CA (1) | CA2953499C (hr) |
| CY (2) | CY1120520T1 (hr) |
| DK (2) | DK3354732T3 (hr) |
| ES (2) | ES2666179T3 (hr) |
| HK (1) | HK1256688A1 (hr) |
| HR (1) | HRP20200523T1 (hr) |
| HU (1) | HUE049405T2 (hr) |
| IL (1) | IL249612B (hr) |
| LT (1) | LT3354732T (hr) |
| MX (2) | MX2016016905A (hr) |
| NZ (1) | NZ765591A (hr) |
| PL (2) | PL3155099T3 (hr) |
| PT (2) | PT3354732T (hr) |
| RS (1) | RS60366B1 (hr) |
| RU (1) | RU2707911C2 (hr) |
| SG (2) | SG10201803444YA (hr) |
| SI (1) | SI3354732T1 (hr) |
| WO (1) | WO2015200334A1 (hr) |
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| US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
| US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US20150165054A1 (en) | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Methods for correcting caspase-9 point mutations |
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