Recleavable Filled-in 5' Overhangs

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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 restriction endonucleases that generate 5´ overhangs which, if filled-in and ligated, result in new restriction sites. 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.

    Restriction enzymes which have degenerate recognition specificities (i.e. recognize more than one sequence) have been excluded from this list. Where isoschizomers exist, only one member of each set is listed. Only commercially available enzymes have been listed.

    Example:

    EcoRI Fragments       XmnI and AseI Sites
     5' ...G  AATTC...3'  Fill-in and Ligate-->  5' ...GATTAATTC...3'
     3' ...CTTAA  G...5'    3' ...CTTAATTAAG...5'

    Table Notes:

    • Enzymes in red have recognition sequences of 6 or 8 bases. Sequence length is indicated by superscript (e.g., AscI8 = 8-base cutter).
    • Enzymes in parenthesis indicate that the new sequence is still a substrate for the original enzyme.
    • A superscript 2 indicates that two identical sites have been generated within the filled-in/ligated sequence. For example, fill-in/ligation of AflII generates the sequence CTTAATTAAG which contains two MseI sites (TTAA).

    | A | B | D | E | F | H | K | M | N | P | S | T | X |           Single Letter Code

    Enzyme  Cleavage Site   After Fill-in/Ligation  Recleaved By
    Acc65I G/GTACC GGTACGTACC BsaAI, HpyCH4IV, RsaI, SnaBI6
    AciI C/CGC CCGCGC (AciI), BstUI, HhaI
    AclI AA/CGTT AACGCGTT AflIII, BstUI, MluI6
    AflII C/TTAAG CTTAATTAAG MseI2, PacI8, Tsp509I
    AgeI A/CCGGT ACCGGCCGGT BsiEI, (BsrFI)2, EaeI, EagI6, HaeIII, HpaII
    ApaLI G/TGCAC GTGCATGCAC Cac8I, NlaIII, NspI, SphI6
    AscI GG/CGCGCC GGCGCGCGCGCC (BssHII)2, BstUI, Cac8I, HhaI
    AvrII C/CTAGG CCTAGCTAGG AluI, (BfaI)2
    BamHI G/GATCC GGATCGATCC AlwI, ClaI6, (DpnII2), TaqαI
    BclI T/GATCA TGATCGATCA ClaI6, (DpnII2), TaqαI
    BfaI C/TAG CTATAG SfcI
    BglII A/GATCT AGATCGATCT ClaI6, DpnII2, TaqαI
    BsiWI C/GTACG CGTACGTACG BsaAI, (BsiWI), HpyCH4IV, RsaI, SnaBI6
    BspDI/ClaI AT/CGAT ATCGCGAT BstUI, NruI6
    BspEI T/CCGGA TCCGGCCGGA BsiEI, EaeI, EagI6, HaeIII, (HpaII)2
    BspHI T/CATGA TCATGCATGA (NlaIII)2, NsiI6
    BsrGI T/GTACA TGTACGTACA BsaAI, (RsaI)2, SnaBI6
    BssHII G/CGCGC GCGCGCGCGC (BssHII), BstUI, Cac8I, HhaI
    BstBI TT/CGAA TTCGCGAA BstUI, NruI6
    DpnII/MboI/Sau3AI /GATC GATCGATC ClaI6, (DpnII), TaqαI
    EagI C/GGCCG CGGCCGGCCG BsiEI, BsrFI, Cac8I, EaeI2, (EagI2), FseI8, HaeIII, HpaII, NaeI
    EcoRI G/AATTC GAATTAATTC AseI6, MseI, Tsp509I, XmnI6
    FatI /CATG CATGCATG BrfBI6, HpyCH4IV, (FatI2)
    HinP1I G/CGC GCGCGC BssHII6, BstUI, Cac8I, (HhaI)
    HindIII A/AGCTT AAGCTAGCTT AluI, BfaI, Cac8I, NheI6
    HpaII/MspI C/CGG CCGCGG AciI, BsaJI, BstUI, BtgI, MspA1I, SacII6
    HpyCH4IV A/CGT ACGCGT AflIII, BstUI, MluI6
    KasI G/GCGCC GGCGCGCGCC (BssHII6)2, (BstUI)2, Cac8I, (HhaI)2
    MfeI C/AATTG CAATTAATTG AseI6, Tsp509I2
    MluI A/CGCGT ACGCGCGCGT BssHII6, BstUI, Cac8I, (HhaI)2
    NarI GG/CGCC GGCGCGCC AscI8, BssHII, BstUI, Cac8I, HhaI
    NcoI C/CATGG CCATGCATGG NlaIII, NsiI6
    NgoMIV G/CCGGC GCCGGCCGGC BsiEI, BsrFI, Cac8I, EaeI, EagI6, HaeIII, HpaII, (NgoMIV2)
    NheI G/CTAGC GCTAGCTAGC AluI, BfaI, Cac8I, (NheI)
    NotI GC/GGCCGC GCGGCCGGCCGC AciI, BsiEI, BsrFI, Cac8I, EaeI, (EagI6)2, Fnu4HI, FseI8, HaeIII, HpaII, NaeI6
    PaeR7I/XhoI C/TCGAG CTCGATCGAG BsiEI, DpnII, PvuI6, (TaqαI)2
    PciI A/CATGT ACATGCATGT HpyCH4V, (NlaIII2), NsiI6
    PspOMI G/GGCCC GGGCCGGCCC BsrFI, Cac8I, FseI8, HaeIII, HpaII, NaeI, Sau96I
    PspXI VC/TCGAGB VCTCGATCGAGB PvuI6, (TaqαI)2
    SalI G/TCGAC GTCGATCGAC BsiEI, DpnII, PvuI6, TaqαI
    SpeI A/CTAGT ACTAGCTAGT AluI, (BfaI)2
    TaqαI T/CGA TCGCGA BstUI, NruI6
    Tsp509I /AATT AATTAATT AseI6, MseI, (Tsp509I2)
    XbaI T/CTAGA TCTAGCTAGA AluI, BfaI
    XmaI C/CCGGG CCCGGCCGGG BsiEI, EaeI, EagI6, HaeIII, HpaII, NciI, ScrFI