Recleavable Filled-in 5' Overhangs

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:

  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.

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


* 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.