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Home  >Technical Resource  > Restriction Endonucleases > Recleavable Blunt Overhangs

Recleavable Blunt 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 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 have been listed

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

Enzyme Ligated to Recleaved by
Afe I
(AGC/GCT)		 BsrB I, MspA1 I (CMG/CGG) Hpa II
BstZ17 I Alu I
EcoR V SfaN I
Sfo I Hae II, Hha I
Fsp I Hha I
Nae I Aci I, Fnu4H I
Sma I Aci I
Alu I
(AG/CT)		 BsrB I, MspA1 I (CMG/CGG) Aci I
BstZ17 I Rsa I
MspA1 I (CMG/CTG), Pvu II Alu I
EcoR V Mbo I
Sfo I Hae III
BsaA I    
(YAC/GTR) BsrB I, MspA1 I (CMG/CGG) Hpa II
(CAC/GTR) Pml I BsaA I, Pml I
(TAC/GTR) Pml I, SnaB I BsaA I
(TAC/GTR) SnaB I BsaA I, SnaB I
BstU I
(CG/CG)		 BsrB I, MspA1 I (CMG/CGG) Aci I, BstU I
BstZ17 I Rsa I
EcoR V Mbo I
Sfo I Hae III
Nru I BstU I
BstZ17 I
(GTA/TAC)		 Alu I, BsrB I, BstU I, Hae III, Msc I, MspA1 I(CMG/CKG), Nru I, Pvu II, Stu I Rsa I
Afe I Alu I
Hinc II (GTY/GAC) Acc I
Ssp I Tsp509 I
Dra I
(TTT/AAA)		 Hinc II (GTY/AAC), Hpa I Mse I
Nru I Taq I
Pme I, Swa I Dra I, Mse I
EcoR V
(GAT/ATC)		 Alu I, BsrB I, BstU I, MspA1 I (CMG/CKG), Pvu II Mbo I
Hae III, Msc I, Stu I Alw I, Mbo I
Afe I, Sfo I, Fsp I SfaN I
Nru I Mbo I, Taq I
Fsp I
(TGC/GCA)		 BsrB I, MspA1 I (CMG/CGG) Hpa II
Afe I, Sfo I Hha I
EcoR V SfaN I
Nae I Aci I, Fnu4H I
Sma I Aci I
Hae III/Pho I
(GG/CC)		 BsrB I, MspA1 I (CMG/CGG) Aci I
BstZ17 I Rsa I
Msc I, Stu I Hae III
EcoR V Alw I, Mbo I
Sfo I Hae III, Asu96 I
Hinc II (GTY/GAC) BsmF I
Hpa I
(GTT/AAC)		 Dra I, Pme I Mse I
Hinc II (GTY/AAC) Hinc II, Hpa I, Mse I
Hinc II (GTY/GAC) Hinc II
Nru I Taq I
Hinc II  
(GTC/RAC) BsrB I, MspA1 I (CMG/CGG) Hpa II
(GTC/RAC) BstZ17 I Acc I
(GTC/RAC) Hae III, Msc I, Stu I BsmF I
(GTT/RAC) Dra I, Pme I Mse I
(GTC/RAC) Hpa I Hinc II
(GTT/RAC) Hpa I Hinc II, Hpa I, Mse I
(GTT/RAC) Nru I Taq I
(GTC/RAC) Rsa I, Sca I Tsp45 I
Msc I
(TGG/CCA)		 BsrB I, MspA1 I (CMG/CGG) Aci I
BstZ17 I Rsa I
Hae III, Stu I Hae III
EcoR V Alw I, Mbo I
Sfo I Hae III, Sau96 I
Hinc II (GTY/GAC) BsmF I
MspA1 I  
(CAG/CKG) Alu I Alu I
(CCG/CKG) Alu I, Hae III, Msc I, Stu I Aci I
(CCG/CKG) BsaA I, Fsp I, Hinc II (GTY/GAC), Pml I, SnaB I Hpa II
(CAG/CKG) BsrB I, Pvu II Aci I, MspA1 I
(CCG/CKG) BsrB I Aci I, BsaJ I, BstU I, MspA1 I, Sac II
(CMG/CKG) BstZ17 I Rsa I
(CCG/CKG) BstU I, Nru I Aci I, BstU I
(CCG/CKG) Afe I Hpa II
(CMG/CKG) EcoR V Mbo I
(CMG/CKG) Sfo I Hae III
(CCG/CKG) Sfo I Hae III, Hpa II
(CCG/CKG) Nae I Hpa II, Nci I, ScrF I
(CAG/CKG) Pvu II Alu I, MspA1 I, Pvu II
(CCG/CKG) Sma I BsaJ I, Hpa II, Nci I, ScrF I
Nae I
(GCC/GGC)		 BsrB I, MspA1 I (CMG/CGG), Sma I Hpa II, Nci I, ScrF I
Afe I, Sfo I, Fsp I Aci I, Fnu4H I
Nru I
(TCG/CGA)		 BsrB I, MspA1 I (CMG/CGG) Aci I, BstU I
BstZ17 I Rsa I
BstU I BstU I
Dra I, Hinc II (GTY/AAC), Hpa I, Pme I, Rsa I, Sca I, Ssp I Taq I
EcoR V Mbo I, Taq I
Sfo I Hae III
Pme I
(GTTT/AAAC)		 Dra I, Swa I Dra I, Mse I
Hinc II (GTY/AAC), Hpa I Mse I
Nru I Taq I
Pml I
(CAC/GTG)		 BsaA I (YAC/GTA), SnaB I BsaA I
BsaA I (YAC/GTG) BsaA I, Pml I
BsrB I, MspA1 I (CMG/CGG) Hpa II
BmgB I Pml I
Pvu II
(CAG/CTG)		 Alu I Alu I
BsrB I, MspA1 I (CMG/CGG) Aci I, MspA1 I
Bstz17 I Rsa I
EcoR V Mbo I
Sfo I Hae III
MspA1 I (CMG/CTG) Alu I, MspA1 I, Pvu II
Rsa I
(GT/AC)		 Hinc II (GTY/GAC) Tsp45 I
Nru I Taq I
Sca I Rsa I
Sca I
(AGT/ACT)		 Hinc II (GTY/GAC) Tsp45 I
Nru I Taq I
Rsa I Rsa I
Sfo I
(GGC/GCC)		 Alu I, BstU I, MspA1 I (CMG/CKG), Nru I, Pvu II Hae III
BsrB I, MspA1 I (CMG/CGG) Hae III, Hpa II
Hae III, Msc I, Stu I Hae III, Sau96 I
Afe I Hae II, Hha I
EcoR V SfaN I
Fsp I Hha I
Nae I Aci I, Fnu4H I
Sma I Aci I
Sma I
(CCC/GGG)		 BsrB I, MspA1 I (CMG/CGG) BsaJ I, Hpa II, Nci I, ScrF I
Afe I, Sfo I, Fsp I Aci I
Nae I Hpa II, Nci I, ScrF I
SnaB I
(TAC/GTA)		 BsaA I (YAC/GTA) BsaA I, SnaB I
BsaA I (YAC/GTG), Pml I BsaA I
BsrB I, MspA1 I (CMG/CGG) Hpa II
Ssp I
(AAT/ATT)		 BstZ17 I Tsp509 I
Nru I Taq I
Stu I
(AGG/CCT)		 BsrB I, MspA1 I (CMG/CGG) Aci I
BstZ17 I Rsa I
Hae III, Msc I Hae III
EcoR V Alw I, Mbo I
Sfo I Hae III, Sau96 I
Hinc II (GTY/GAC) BsmF I
Swa I
(ATTT/AAAT)		 Dra I, Pme I Dra I, Mse I