Compatible Cohesive Ends and Generation of New Restriction Sites
New restriction sites can be generated by ligation of DNA fragments with compatible cohesive or blunt ends. These new restriction sites may be generated by:
- Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
- Cleavage with two restriction endonucleases that produce blunt ends.
- Cleavage with two restriction endonucleases that produce compatible overhangs.
Restriction endonucleases that produce compatible cohesive ends often produce recleavable ligation products. The combinations listed were identified by computer analysis, and although we have tried to ensure their accuracy, they have not necessarily been confirmed by experimentation.
Where isoschizomers exist, only one member of each set is listed. Only enzymes available from New England Biolabs have been listed.
Enzymes that have degenerate recognition sequences (i.e. recognize more than one sequence) are followed by a specific sequence in parentheses and are only listed if a non-degenerate equivalent does not exist. Be aware that these degenerate enzymes will cleave sequences in addition to the one listed.
A "--" denotes a ligation product that cannot be recleaved.
| A | B | C | D | E | H | K | M | N | P | R | S | T | X | single letter code
| Enzyme | Ligated To | Recleaved By |
|---|---|---|
| Acc65I (G/GTACC) |
BanI (G/GTACC) | Acc65I, BanI, KpnI, NlaIV, RsaI |
| BsiWI, BsrGI | RsaI | |
| AccI | |
|
| (GT/CGAC) | AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI | -- |
| (GT/CGAC) | ClaI, BstBI, TaqI-v2 | TaqI-v2 |
| AciI (C/CGC) |
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI-v2 | -- |
| BsaHI (GR/CGCC), HinP1I, NarI | AciI | |
| HpaII | HpaII | |
| AclI (AA/CGTT) |
AccI (GT/CGAC), AciI, ClaI, BstBI, HinP1I, HpaII, NarI, TaqI-v2 | -- |
| AgeI * (A/CCGGT) |
AvaI (C/CCGGG), XmaI | HpaII, NciI, ScrFI |
| BsaWI, BspEI | BsaWI, HpaII | |
| BsrFI (A/CCGGT), SgrAI (CA/CCGGTG) | AgeI, BsaWI, BsrFI, HpaII | |
| NgoMIV | BsrFI, HpaII | |
| ApaI (GGGCC/C) |
BanII (GGGCC/C), Bsp1286I (GGGCC/C) | ApaI, BanII, Bsp120I, Bsp1286I, HaeIII, NlaIV, Sau96I |
| ApaLI (G/TGCAC) |
SfcI (C/TGCAG) | BsgI |
| ApoI | |
|
| (A/AATTY) | EcoRI | ApoI, Tsp509I |
| (G/AATTY) | EcoRI | ApoI, EcoRI, Tsp509I |
| (R/AATTY) | MfeI, Tsp509I | Tsp509I |
| AscI (GG/CGCGCC) |
AflIII (A/CGCGT), MluI | BstUI, HhaI |
| BssHII | BssHII, BstUI, Cac8I, HhaI | |
| AseI (AT/TAAT) |
BfaI, Csp6I, NdeI | -- |
| MseI | MseI | |
| AsiSI (GCGAT/CGC) |
BsiEI (CGAT/CG) | DpnII, PvuI |
| PacI | MseI | |
| PvuI | DpnII, PvuI | |
| AvaI | |
|
| (C/CCGGG) | AgeI, BsaWI, BspEI, BsrFI (R/CCGGY), NgoMIV, SgrAI (CR/CCGGYG) | HpaII, NciI, ScrFI |
| (C/TCGAG) | XhoI | AvaI, TaqI-v2, XhoI |
| (C/TCGAG) | SalI | TaqI-v2 |
| (C/CCGGG) | XmaI | AvaI, BsaJI, HpaII, NciI, ScrFI, SmaI |
| AvaII (G/GWCC) |
PpuMI (RG/GACCY) | AvaII, NlaIV, Sau96I |
| RsrII | AvaII, Sau96I | |
| PpuMI (RG/GTCCY) | AvaII, BsmFI, NlaIV, Sau96I | |
| AvrII (C/CTAGG) |
NheI, SpeI, XbaI | BfaI |
| StyI (C/CTAGG) | AvrII, BfaI, BsaJI, StyI | |
| BamHI * (G/GATCC) |
BclI, DpnII | AlwI, DpnII |
| BglII, BstYI (R/GATCY) | AlwI, BstYI, DpnII | |
| BstYI (G/GATCC) | AlwI, BamHI, BstYI, DpnII, NlaIV | |
| BanI | |
|
| (G/GTACC) | Acc65I | Acc65I, BanI, KpnI, NlaIV, RsaI |
| (G/GCGCC) | KasI | BanI, BsaHI, HaeII, HhaI, KasI, NarI, NlaIV |
| (G/GTACC) | BsiWI, BsrGI | RsaI |
| BanII | |
|
| (GGGCC/C) | ApaI, Bsp1286I (GGGCC/C) | ApaI, BanII, Bsp1286I, HaeIII, NlaIV, Sau96I |
| (GAGCT/C) | Bsp1286I (GAGCT/C), SacI | AluI, BanII, BsiHKAI, Bsp1286I, SacI |
| BclI * (T/GATCA) |
BamHI, BstYI (R/GATCY) | AlwI, DpnII |
| BglII, MboI | DpnII | |
| BfaI (C/TAG) |
AseI, Csp6I, MseI, NdeI | -- |
| BglII (A/GATCT) |
BamHI, BstYI (R/GATCY) | AlwI, BstYI, DpnII |
| BclI, DpnII | DpnII | |
| BsaHI | |
|
| (GR/CGYC) | AccI (GT/CGAC), ClaI, BstBI, TaqI-v2 | -- |
| (GA/CGYC) | AciI, HinP1I | HgaI |
| (GG/CGYC) | AciI, HinP1I | HhaI |
| (GG/CGYC) | HpaII | AciI |
| (GA/CGYC) | NarI | BsaHI, HgaI |
| (GG/CGYC) | NarI | BanI, BsaHI, HaeII, HhaI, NarI, NlaIV |
| BsaWI (W/CCGGW) |
AgeI, BsrFI (R/CCGGY), SgrAI (CR/CCGGYG) | AgeI, BsaWI, BsrFI, HpaII |
| AvaI (C/CCGGG), XmaI | HpaII, NciI, ScrFI | |
| BspEI | BsaWI, BspEI, HpaII | |
| BsrFI (R/CCGGY), NgoMIV | BsrFI, HpaII | |
| NgoMIV | HpaII | |
| BsiEI | |
|
| (CGAT/CG) | PacI | MseI |
| (CGAT/CG) | PvuI | BsiEI, DpnII, PvuI |
| (CGGC/CG) | SacII | AciI |
| BsiHKAI (GTGCA/C) |
Bsp1286I (GTGCA/C) | BsiHKAI, Bsp1286I |
| Bsp1286I (GAGCA/C) | BsiHKAI, Bsp1286I | |
| Bsp1286I (GAGCT/C), SacI | AluI, BanII, BsiHKAI, Bsp1286I, SacI | |
| NsiI | -- | |
| PstI, SbfI | BsgI | |
| BsiWI (C/GTACG) |
Acc65I, BanI (G/GTACC), BsrGI | RsaI |
| Bsp1286I | |
|
| (GGGCC/C) | ApaI, BanII (GGGCC/C) | ApaI, BanII, Bsp1286I, HaeIII, NlaIV, Sau96I |
| (GTGCA/C) | BsiHKAI | ApaLI, BsiHKAI, Bsp1286I |
| (GGGCC/C) | BanII (GGGCC/C) | BanII, Bsp1286I |
| (GAGCT/C) | BanII (GAGCT/C), BsiHKAI, SacI | AluI, BanII, BsiHKAI, Bsp1286I, SacI |
| (GWGCW/C) | BsiHKAI | BsiHKAI, Bsp1286I |
| (GTGCA/C) | NsiI | -- |
| (GTGCA/C) | PstI, SbfI | BsgI |
| BspEI (T/CCGGA) |
AgeI, BsaWI, BsrFI (R/CCGGY), SgrAI (CR/CCGGYG) | BsaWI, HpaII |
| AvaI (C/CCGGG), XmaI | HpaII, NciI, ScrFI | |
| BsaWI | BsaWI, BspEI, HpaII | |
| BsrFI (R/CCGGY), NgoMIV | HpaII | |
| BspHI (T/CATGA) |
FatI, NcoI, PciI | FatI, NlaIII |
| BsrFI | |
|
| (A/CCGGY) | AgeI, BsaWI | AgeI, BsaWI, BsrFI, HpaII |
| (G/CCGGY) | AgeI, BsaWI, NgoMIV | BsrFI, HpaII |
| (R/CCGGY) | AvaI (C/CCGGG), XmaI | HpaII, NciI, ScrFI |
| (A/CCGGY) | BsaWI, BspEI | BsaWI, HpaII |
| (R/CCGGY) | BsaWI, BspEI | HpaII |
| (G/CCGGY) | NgoMIV | BsrFI, Cac8I, HpaII, NaeI |
| (CR/CCGGYG) | SgrAI | BsrFI, HpaII |
| BsrGI (T/GTACA) |
Acc65I, BanI (G/GTACC), BsiWI | RsaI |
| BssHII (G/CGCGC) |
MluI | BstUI, HhaI |
| AscI | BssHII, BstUI, Cac8I, HhaI | |
| BstBI (TT/CGAA) |
AccI (GT/CGAC), ClaI, TaqI-v2 | TaqI-v2 |
| AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI | -- | |
| BstYI | |
|
| (A/GATCY) | BamHI, BglII | AlwI, BstYI, DpnII |
| (G/GATCY) | BamHI | AlwI, BamHI, BstYI, DpnII, NlaIV |
| (R/GATCY) | BclI, DpnII | DpnII |
| (G/GATCY) | BclI, DpnII | AlwI, DpnII |
| (A/GATCY) | BglII | BglII, BstYI, DpnII |
| ClaI (AT/CGAT) |
AccI (GT/CGAC), BstBI, TaqI-v2 | TaqI-v2 |
| AciI, AclI, BsaHI (GR/CGYC), HinP1I, HpaII, NarI | -- | |
| DpnII/MboI/ Sau3AI (/GATC) |
BamHI, BstYI (R/GATCC) | AlwI, DpnII |
| BclI, BglII, BstYI (R/GATCY) | DpnII | |
| EaeI | |
|
| (Y/GGCCR) | PspOMI | HaeIII, Sau96I |
| (C/GGCCR) | EagI | BsiEI, EaeI, EagI, HaeIII |
| (T/GGCCR) | EagI | EaeI, HaeIII |
| (C/GGCCR) | NotI | AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII |
| (T/GGCCR) | NotI | AciI, EaeI, Fnu4HI, HaeIII |
| EagI * (C/GGCCG) |
PspOMI | HaeIII, Sau96I |
| EaeI (Y/GGCCR) | EaeI, HaeIII | |
| EaeI (C/GGCCG) | BsiEI, EaeI, EagI, HaeIII | |
| NotI | AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII | |
| EcoRI * (G/AATTC) |
ApoI (G/AATTC) | ApoI, EcoRI, Tsp509I |
| ApoI (R/AATTY) | ApoI, Tsp509I | |
| MfeI, Tsp509I | Tsp509I | |
| FatI (/CATG) |
BspHI, NcoI, PciI | FatI, NlaIII |
| HinP1I (G/CGC) |
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI-v2 | -- |
| AciI, BsaHI (GR/CGCC), NarI | HhaI | |
| BsaHI (GR/CGTC) | HgaI | |
| HpaII | AciI | |
| HpaII/MspI (C/CGG) |
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI-v2 | -- |
| AciI, BsaHI (GR/CGCC), HinP1I, NarI | AciI | |
| KasI (G/GCGCC) |
BanI (G/GCGCC) | BanI, BsaHI, HaeII, HhaI, KasI, NarI, NlaIV |
| MfeI * (C/AATTG) |
ApoI (R/ATTTY), EcoRI, Tsp509I | Tsp509I |
| MluI (A/CGCGT) |
AscI, BssHII | BstUI, HhaI |
| MseI (T/TAA) |
AseI | MseI |
| BfaI, Csp6I, NdeI | -- | |
| NarI (GG/CGCC) |
AccI (GT/CGAC), AclI, ClaI, BstBI, TaqI-v2 | -- |
| AciI, HinP1I | HhaI | |
| BsaHI (GR/CGCC) | BanI, BsaHI, HaeII, HhaI, NarI, NlaIV | |
| BsaHI (GR/CGTC) | BsaHI, HgaI | |
| HpaII | AciI | |
| NcoI * (C/CATGG) |
BspHI, FatI, PciI | FatI, NlaIII |
| NdeI (CA/TATG) |
AseI, BfaI, Csp6I, MseI | -- |
| NgoMIV (G/CCGGC) |
AgeI, BsaWI, BsrFI (R/CCGGY), SgrAI | BsrFI, HpaII |
| AvaI (C/CCGGG), XmaI | HpaII, NciI, ScrFI | |
| BsaWI, BspEI | HpaII | |
| BsrFI (R/CCGGC), SgrAI | BsrFI, Cac8I, HpaII, NaeI | |
| NheI * (G/CTAGC) |
AvrII, SpeI, StyI (C/CTAGG), XbaI | BfaI |
| NlaIII (CATG/) |
SphI, NspI | NlaIII |
| NotI * (GC/GGCCGC) |
PspOMI | AciI, EaeI, Fnu4HI, HaeIII |
| EagI | AciI, BsiEI, EaeI, EagI, Fnu4HI, HaeIII | |
| EaeI (Y/GGCCR) | AciI, BsiEI, EaeI, Fnu4HI, HaeIII | |
| NsiI (ATGCA/T) |
BsiHKAI (GTGCA/C), Bsp1286I (GTGCA/C), PstI, SbfI | -- |
| NspI (RCATG/Y) |
NlaIII, SphI | NlaIII,NspI |
| PacI (TTAAT/TAA) |
AsiSI, BsiEI (CGAT/CG), PvuI | MseI |
| PciI (A/CATGT) |
BspHI, FatI, NcoI | FatI, NlaIII |
| PpuMI | |
|
| (RG/GWCCY) | AvaII, RsrII | AvaII, Sau96I |
| (GG/GTCCY) | AvaII, RsrII | AvaII, BsmFI, NlaIV, Sau96I |
| (GG/GACCY) | AvaII, RsrII | AvaII, NlaIV, Sau96I |
| PspOMI (G/GGCCC) |
EaeI (Y/GGCCR), EagI | HaeIII, Sau96I |
| NotI | AciI, Fnu4HI, HaeIII, Sau96I | |
| PspXI (VC/TCGAGB) |
XhoI, TliI | XhoI, TliI |
| SalI | TaqI-v2 | |
| PstI * (CTGCA/G) |
BsiHKAI, Bsp1286I (GTGCA/C) | BsgI |
| NsiI | -- | |
| SbfI | PstI | |
| PvuI * (CGAT/CG) |
AsiSI | DpnI, PvuI |
| PacI | MseI | |
| BsiEI (CGAT/CG) | BsiEI, DpnII, PvuI | |
| RsrII (CG/GWCCG) |
AvaII, PpuMI (RG/GACCY) | AvaII, Sau96I |
| PpuMI (RG/GACCY) | AvaII, NlaIV, Sau96I | |
| PpuMI (RG/GTCCY) | AvaII, BsmFI, NlaIV, Sau96I | |
| SacI * (GAGCT/C) |
BanII (GAGCT/C), BsiHKAI, Bsp1286I (GAGCT/C) | AluI, BanII, BsiHKAI, Bsp1286I, SacI |
| SacII (CCGC/GG) |
BsiEI (CGGC/CG) | AciI |
| SalI * (G/TCGAC) |
PspXI, XhoI | TaqI-v2 |
| SbfI * (CCTGCA/GG) |
BsiHKAI, Bsp1286I (GTGCA/C) | BsgI |
| NsiI | -- | |
| PstI | PstI | |
| SfcI (C/TGCAG) |
ApaLI | BsgI |
| SgrAI (CR/CCGGYG) |
See BsrFI | |
| SpeI * (A/CTAGT) |
AvrII, NheI, StyI (C/CTAGG), XbaI | BfaI |
| SphI * (GCATG/C) |
NlaIII, NspI | NlaIII, NspI |
| StyI * | AvrII | AvrII, BfaI, BsaJI, StyI |
| (C/CTAGG) | NheI, SpeI, XbaI | BfaI |
| (C/CATGG) | BspHI | NlaIII |
| NcoI | BsaJI, NcoI, NlaIII, StyI | |
| TaqI-v2 (T/CGA) |
AccI (GT/CGAC), ClaI, BstBI | TaqI-v2 |
| AciI, AclI, BsaHI (GR/CGYC), HinP1I,HpaII, NarI | -- | |
| Tsp509I (/AATT) |
ApoI (R/AATTY), EcoRI, MfeI | Tsp509I |
| XbaI (T/CTAGA) |
AvrII, NheI, SpeI, StyI (C/CTAGG) | BfaI |
| XhoI/TliI (C/TCGAG) |
PspXI | XhoI, TliI |
| SalI | TaqI-v2 | |
| XmaI (C/CCGGG) |
AgeI, BsaWI, BspEI, BsrFI, NgoMIV, SgrAI | HpaII, NciI, ScrFI |
| AvaI (C/CCGGG) | AvaI, BsaJI, HpaII, NciI, ScrFI, SmaI, XmaI |
* HF (high fidelity) versions of these enzymes are available.
