Restriction enzymes for ARRF

Here is a list of restriction enzymes that could be used for the ARRF technique. Whether a particular enzyme cuts rarely enough to be suitable depends on the genome size and GC content of your organism; most of the six-cutters listed here will cut too frequently to be useful for the ARRF technique in most plants and animals. Six-cutters would be useful for prokaryotes and those eukaryotes with small genomes, and those six-cutters with all GCs in their restriction sites may be useful for eukaryotes with normal-sized genomes that are AT-rich.

Enzymes that leave 5' overhangs are listed first, because you can buy a single (expensive) labeled oligo and pair it with a variety of (inexpensive) unlabeled oligos to create adapters to match 5' overhangs. I have attempted to list all of the commercially available six-cutters, seven-cutters and eight-cutters that leave 5' overhangs. Enzymes that leave 3' overhangs require a different labeled oligo for each overhang, so I have listed only eight-cutters and the six-cutters that leave the same overhangs. For more information about these enzymes, including where to buy them, see REBASE.

Enzymes that are grouped together leave the same cohesive end. When more than one name is shown for a restriction sequence, more than one isoschizomer is commercially available.

Enzymes that leave 5' overhangs:

AscI                 GG^CGCG_CC (eight-cutter)
BsePI, BssHII        G^CGCG_C   (all GC)
MluI                 A^CGCG_T

NotI, CciNI          GC^GGCC_GC (eight-cutter)
EagI, XmaIII         C^GGCC_G   (all GC)
PspOMI               G^GGCC_C   (all GC)

SgrAI                CR^CCGG_YG (seven-cutter)
AgeI                 A^CCGG_T
BspEI                T^CCGG_A
NgoMIV               G^CCGG_C   (all GC)
XmaI                 C^CCGG_G   (all GC)

BbvCI                CC^TCA_GC  (seven-cutter)
 
AvrII                C^CTAG_G
NheI                 G^CTAG_C
SpeI                 A^CTAG_T
XbaI                 T^CTAG_A

AclI                 AA^CG_TT
AsuII, BstBI         TT^CG_AA
ClaI                 AT^CG_AT
NarI                 GG^CG_CC   (all GC)

BspHI                T^CATG_A
BspLU11I, PciI       A^CATG_T
NcoI                 C^CATG_G

BamHI                G^GATC_C
BclI                 T^GATC_A
BglII                A^GATC_T

Acc65I               G^GTAC_C
BsiWI                C^GTAC_G
Bsp1407I, BsrGI      T^GTAC_A

EcoRI                G^AATT_C
MfeI                 C^AATT_G

AseI, VspI           AT^TA_AT
NdeI                 CA^TA_TG

SalI                 G^TCGA_C
XhoI                 C^TCGA_G

ApaLI                G^TGCA_C
Ppu10I               A^TGCA_T

BssSI                C^ACGA_G

HindIII              A^AGCT_T

KasI                 G^GCGC_C   (all GC)

AflII                C^TTAA_G

Enzymes that leave 3' overhangs:

PacI                 TTA_AT^TAA (eight-cutter)
AsiSI, SgfI          GCG_AT^CGC (eight-cutter)
PvuI                 CG_AT^CG

SbfI, Sse8387I, SdaI CC_TGCA^GG (eight-cutter)
NsiI                 A_TGCA^T
PstI                 C_TGCA^G

FseI                 GG_CCGG^CC (eight-cutter)

Enzymes that leave multiple overhangs:

These enzymes, if used with an adapter that matched just one of the possible cohesive ends, might be used as seven-cutters, nine-cutters, or even an eleven-cutter (SfiI). I haven't tried this yet, though.

BlpI                 GC^TNA_GC
BstEII               G^GTNAC_C
Bsu36I               CC^TNA_GG
RsrII                CG^GWC_CG
SanDI                GG^GWC_CC
SexAI                A^CCWGG_T

AlwNI                CAG_NNN^CTG
BglI                 GCCN_NNN^NGGC
DraIII               CAC_NNN^GTG
PflMI                CCAN_NNN^NTGG

SfiI                 GGCCN_NNN^NGGCC

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Send comments to John H. McDonald (mcdonald@udel.edu).

This page was last revised July 23, 2002. Its URL is http://udel.edu/~mcdonald/enzymes.html