Recleavable Blunt Ends

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  • New restriction sites can be generated by ligation of DNA fragments with compatible ends. These ends may be generated by:

    1. Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
    2. Cleavage with two restriction endonucleases that produce blunt ends.
    3. Cleavage with two restriction endonucleases that produce compatible overhangs.

    Compatible ends, generated by each of the above methods, can be ligated to produce DNA sequences that often contain useful restriction endonuclease sites. Generation of these sites is detailed in the following tables.

    The table below lists combinations of blunt-end restriction endonucleases that 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.

    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. Where isoschizomers exist, only one member of each set is listed. Only commercially available enzymes are shown.

    | A | B | D | E | F | H | M | N | P | R | S |    single letter code

    Enzyme Ligated to Recleaved by
    AfeI (AGC/GCT) BsrBI, MspA1I (CMG/CGG) HpaII
    BstZ17I AluI
    EcoRV SfaNI
    SfoI HaeII, HhaI
    FspI HhaI
    NaeI AciI, Fnu4HI
    SmaI AciI
    AluI (AG/CT) BsrBI, MspA1I (CMG/CGG) AciI
    BstZ17I RsaI
    MspA1I (CMG/CTG), PvuII AluI
    EcoRV MboI
    SfoI HaeIII
    BsaAI    
    (YAC/GTR) BsrBI, MspA1I (CMG/CGG) HpaII
    (CAC/GTR) PmlI BsaAI, PmlI
    (TAC/GTR) PmlI, SnaBI BsaAI
    (TAC/GTR) SnaBI BsaAI, SnaBI
    BstUI (CG/CG) BsrBI, MspA1I (CMG/CGG) AciI, BstUI
    BstZ17I RsaI
    EcoRV MboI
    SfoI HaeIII
    NruI BstUI
    BstZ17I (GTA/TAC) AluI, BsrBI, BstUI, HaeIII, MscI, MspA1I(CMG/CKG), NruI, PvuII, StuI RsaI
    AfeI AluI
    HincII (GTY/GAC) AccI
    SspI Tsp509I
    DraI (TTT/AAA) HincII (GTY/AAC), HpaI MseI
    NruI TaqαI
    PmeI, SwaI DraI, MseI
    EcoRV *+ 
    (GAT/ATC)
    AluI, BsrBI, BstUI, MspA1I (CMG/CKG), PvuII MboI
    HaeIII, MscI, StuI AlwI, MboI
    AfeI, SfoI, FspI SfaNI
    NruI MboI, TaqαI
    FspI (TGC/GCA) BsrBI, MspA1I (CMG/CGG) HpaII
    AfeI, SfoI HhaI
    EcoRV SfaNI
    NaeI AciI, Fnu4HI
    SmaI AciI
    HaeIII/PhoI (GG/CC) BsrBI, MspA1I (CMG/CGG) AciI
    BstZ17I RsaI
    MscI, StuI HaeIII
    EcoRV AlwI, MboI
    SfoI HaeIII, Asu96I
    HincII (GTY/GAC) BsmFI
    HpaI (GTT/AAC) DraI, PmeI MseI
    HincII (GTY/AAC) HincII, HpaI, MseI
    HincII (GTY/GAC) HincII
    NruI TaqαI
    HincII
    (GTC/RAC) BsrBI, MspA1I (CMG/CGG) HpaII
    (GTC/RAC) BstZ17I AccI
    (GTC/RAC) HaeIII, MscI, StuI BsmFI
    (GTT/RAC) DraI, PmeI MseI
    (GTC/RAC) HpaI HincII
    (GTT/RAC) HpaI HincII, HpaI, MseI
    (GTT/RAC) NruI TaqαI
    (GTC/RAC) RsaI, ScaI Tsp45I
    MscI (TGG/CCA) BsrBI, MspA1I (CMG/CGG) AciI
    BstZ17I RsaI
    HaeIII, StuI HaeIII
    EcoRV AlwI, MboI
    SfoI HaeIII, Sau96I
    HincII (GTY/GAC) BsmFI
    MspA1I
    (CAG/CKG) AluI AluI
    (CCG/CKG) AluI, HaeIII, MscI, StuI AciI
    (CCG/CKG) BsaAI, FspI, HincII (GTY/GAC), PmlI, SnaBI HpaII
    (CAG/CKG) BsrBI, PvuII AciI, MspA1I
    (CCG/CKG) BsrBI AciI, BsaJI, BstUI, MspA1I, SacII
    (CMG/CKG) BstZ17I RsaI
    (CCG/CKG) BstUI, NruI AciI, BstUI
    (CCG/CKG) AfeI HpaII
    (CMG/CKG) EcoRV MboI
    (CMG/CKG) SfoI HaeIII
    (CCG/CKG) SfoI HaeIII, HpaII
    (CCG/CKG) NaeI HpaII, NciI, ScrFI
    (CAG/CKG) PvuII AluI, MspA1I, PvuII
    (CCG/CKG) SmaI BsaJI, HpaII, NciI, ScrFI
    NaeI (GCC/GGC) BsrBI, MspA1I (CMG/CGG), SmaI HpaII, NciI, ScrFI
    AfeI, SfoI, FspI AciI, Fnu4HI
    NruI (TCG/CGA) BsrBI, MspA1I (CMG/CGG) AciI, BstUI
    BstZ17I RsaI
    BstUI BstUI
    DraI, HincII (GTY/AAC), HpaI, PmeI, RsaI, ScaI, SspI TaqαI
    EcoRV MboI, TaqαI
    SfoI HaeIII
    PmeI (GTTT/AAAC) DraI, SwaI DraI, MseI
    HincII (GTY/AAC), HpaI MseI
    NruI TaqαI
    PmlI (CAC/GTG) BsaAI (YAC/GTA), SnaBI BsaAI
    BsaAI (YAC/GTG) BsaAI, PmlI
    BsrBI, MspA1I (CMG/CGG) HpaII
    BmgBI PmlI
    PvuII * (CAG/CTG) AluI AluI
    BsrBI, MspA1I (CMG/CGG) AciI, MspA1I
    Bstz17I RsaI
    EcoRV MboI
    SfoI HaeIII
    MspA1I (CMG/CTG) AluI, MspA1I, PvuII
    RsaI (GT/AC) HincII (GTY/GAC) Tsp45I
    NruI TaqαI
    ScaI RsaI
    ScaI*+
     (AGT/ACT)
    HincII (GTY/GAC) Tsp45I
    NruI TaqαI
    RsaI RsaI
    SfoI (GGC/GCC) AluI, BstUI, MspA1I (CMG/CKG), NruI, PvuII HaeIII
    BsrBI, MspA1I (CMG/CGG) HaeIII, HpaII
    HaeIII, MscI, StuI HaeIII, Sau96I
    AfeI HaeII, HhaI
    EcoRV SfaNI
    FspI HhaI
    NaeI AciI, Fnu4HI
    SmaI AciI
    SmaI (CCC/GGG) BsrBI, MspA1I (CMG/CGG) BsaJI, HpaII, NciI, ScrFI
    AfeI, SfoI, FspI AciI
    NaeI HpaII, NciI, ScrFI
    SnaBI (TAC/GTA) BsaAI (YAC/GTA) BsaAI, SnaBI
    BsaAI (YAC/GTG), PmlI BsaAI
    BsrBI, MspA1I (CMG/CGG) HpaII
    SspI*+
     (AAT/ATT)
    BstZ17I Tsp509I
    NruI TaqαI
    StuI (AGG/CCT) BsrBI, MspA1I (CMG/CGG) AciI
    BstZ17I RsaI
    HaeIII, MscI HaeIII
    EcoRV AlwI, MboI
    SfoI HaeIII, Sau96I
    HincII (GTY/GAC) BsmFI
    SwaI (ATTT/AAAT) DraI, PmeI DraI, MseI


    * HF (high fidelity) versions of these enzymes are available for simplified reactions and reduced star activity, at no additional cost.

    + A RE-Mix version of this, or the HF version of this enzyme is available.