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Home  >Technical Resource  > Restriction Endonucleases > Recleavable Filled-in 5' Overhangs

Recleavable Filled-in 5' Overhangs

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:  EcoR I Fragments      XmnI and AseI Sites
5´ ...G
 AATTC...3´
      Fill-in and Ligate-->       5´ ...GAATTAATTC...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., Asc I8 = 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 Afl II generates the sequence CTTAATTAAG which contains two Mse I 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
Acc65 I G/GTACC GGTACGTACC BsaA I, HpyCH4 IV, Rsa I, SnaB I6
Aci I C/CGC CCGCGC (Aci I), BstU I, Hha I
Acl I AA/CGTT AACGCGTT Afl III, BstU I, Mlu I6
Afl II C/TTAAG CTTAATTAAG Mse I2, Pac I8, Tsp509 I
Age I A/CCGGT ACCGGCCGGT BsiE I, (BsrF I)2, Eae I, Eag I6, Hae III, Hpa II
ApaL I G/TGCAC GTGCATGCAC Cac8 I, Nla III, Nsp I, Sph I6
Asc I GG/CGCGCC GGCGCGCGCGCC (BssH II)2, BstU I, Cac8 I, Hha I
Avr II C/CTAGG CCTAGCTAGG Alu I, (Bfa I)2
BamH I G/GATCC GGATCGATCC Alw I, Cla I6, (Dpn II2), Taq I
Bcl I T/GATCA TGATCGATCA Cla I6, (Dpn II2), Taq I
Bfa I C/TAG CTATAG Sfc I
Bgl II A/GATCT AGATCGATCT Cla I6, Dpn II2, Taq I
BsiW I C/GTACG CGTACGTACG BsaA I, (BsiW I), HpyCH4 IV, Rsa I, SnaB I6
BspD I/Cla I AT/CGAT ATCGCGAT BstU I, Nru I6
BspE I T/CCGGA TCCGGCCGGA BsiE I, Eae I, Eag I6, Hae III, (Hpa II)2
BspH I T/CATGA TCATGCATGA (Nla III)2, Nsi I6
BsrG I T/GTACA TGTACGTACA BsaA I, (Rsa I)2, SnaB I6
BssH II G/CGCGC GCGCGCGCGC (BssH II), BstU I, Cac8 I, Hha I
BstB I TT/CGAA TTCGCGAA BstU I, Nru I6
Dpn II/Mbo I/Sau3A I /GATC GATCGATC Cla I6, (Dpn II), Taq I
Eag I C/GGCCG CGGCCGGCCG BsiE I, BsrF I, Cac8 I, Eae I2, (Eag I2), Fse I8, Hae III, Hpa II, Nae I
EcoR I G/AATTC GAATTAATTC Ase I6, Mse I, Tsp509 I, Xmn I6
Fat I /CATG CATGCATG BrfB I6, HpyCH4 IV, (Fat I2)
HinP1 I G/CGC GCGCGC BssH II6, BstU I, Cac8 I, (Hha I)
Hind III A/AGCTT AAGCTAGCTT Alu I, Bfa I, Cac8 I, Nhe I6
Hpa II/Msp I C/CGG CCGCGG Aci I, BsaJ I, BstU I, Btg I, MspA1 I, Sac II6
HpyCH4 IV A/CGT ACGCGT Afl III, BstU I, Mlu I6
Kas I G/GCGCC GGCGCGCGCC (BssH II6)2, (BstU I)2, Cac8 I, (Hha I)2
Mfe I C/AATTG CAATTAATTG Ase I6, Tsp509 I2
Mlu I A/CGCGT ACGCGCGCGT BssH II6, BstU I, Cac8 I, (Hha I)2
Nar I GG/CGCC GGCGCGCC Asc I8, BssH II, BstU I, Cac8 I, Hha I
Nco I C/CATGG CCATGCATGG Nla III, Nsi I6
NgoM IV G/CCGGC GCCGGCCGGC BsiE I, BsrF I, Cac8 I, Eae I, Eag I6, Hae III, Hpa II, (NgoM IV2)
Nhe I G/CTAGC GCTAGCTAGC Alu I, Bfa I, Cac8 I, (Nhe I)
Not I GC/GGCCGC GCGGCCGGCCGC Aci I, BsiE I, BsrF I, Cac8 I, Eae I, (Eag I6)2, Fnu4H I, Fse I8, Hae III, Hpa II, Nae I6
PaeR7 I/Xho I C/TCGAG CTCGATCGAG BsiE I, Dpn II, Pvu I6, (Taq I2)
Pci I A/CATGT ACATGCATGT HpyCH4 V, (Nla III2), Nsi I6
PspOM I G/GGCCC GGGCCGGCCC BsrF I, Cac8 I, Fse I6, Hae III, Hpa II, Nae I, Sau96 I
PspX I VC/TCGAGB VCTCGATCGAGB Pvu I6, (Taq I2)
Sal I G/TCGAC GTCGATCGAC BsiE I, Dpn II, Pvu I6, Taq I
Spe I A/CTAGT ACTAGCTAGT Alu I, (Bfa I)2
Taq I T/CGA TCGCGA BstU I, Nru I6
Tsp509 I /AATT AATTAATT Ase I6, Mse I, (Tsp509 I2)
Xba I T/CTAGA TCTAGCTAGA Alu I, Bfa I
Xma I C/CCGGG CCCGGCCGGG BsiE I, Eae I, Eag I6, Hae III, Hpa II, Nci I, ScrF I