Some restriction enzymes cleave DNA poorly when their recognition sites are located close to DNA termini. Table below lists the activities of Fermentas restriction enzymes when their target sites are located close to the end of a PCR product.

Cleavage Efficiency Close to the Termini of PCR Fragments

Experiments were performed as follows:
PCR primers were designed with 1-5 extra nucleotides at their 5’-end adjacent to the recognition site for the restriction enzyme. The 5’-end was labeled with [³²P] by T4 Polynucleotide Kinase and these labeled primers were used in the PCR reaction. PCR products were purified with the Silica Bead DNA Gel Extraction Kit and precipitated with ethanol. The PCR products (0.5 µg) were incubated with 10 units of restriction enzymes in the optimal buffer for 1 hour at the recommended temperature. Reaction products were separated on 10% PAGE and the cleavage efficiency was determined using OptiQuant® Image Analysis software.

Note

Incubation with NdeI was performed for 16 hours.

Table. Cleavage Efficiency Close to the Termini of PCR Fragments

Enzyme	Bp from the recognition site to fragment end
	1	2	3	4	5
AanI (PsiI)	0	0-20	20-50
AarI	20-50		50-100
AasI (DrdI)	50-100
AatII	0	0-20	20-50	50-100
Acc65I (Asp718I)	0-20	50-100
AdeI (DraIII)	50-100
AjiI (BmgBI)	50-100
AluI	0-20	20-50	50-100
Alw21I (BsiHKAI)	50-100
Alw26I	50-100
Alw44I	0	20-50	50-100
ApaI	50-100
BamHI	50-100
BauI (BssSI)	0-20	20-50	50-100
BclI	0	50-100
BcnI (NciI)	20-50		50-100
BcuI (SpeI)	50-100
BfiI (BmrI)	50-100
BfmI (SfcI)	50-100
BfuI (BciVI)	50-100
BglI	20-50	50-100
BglII	0	50-100
Bme1390I (ScrFI)	20-50		50-100
BoxI (PshAI)	0	50-100
BpiI (BbsI)	50-100
Bpu10I	20-50	50-100
Bpu1102I (BlpI)	50-100
BseDI (BsaJI)	0	50-100
BseGI (BtsCI)	50-100
BseJI (BsaBI)	0	50-100
BseLI (BslI)	0	50-100
BseMI (BsrDI)	0-20	50-100
BseMII (BspCNI)	50-100
BseNI (BsrI)	0	50-100
BseSI (Bme1580I)	50-100
BseXI (BbvI)	20-50	50-100
Bsh1236I (BstUI)	50-100
Bsh1285I (BsiEI)	0-20	50-100
BshNI (BanI)	50-100
BshTI (AgeI)	20-50	50-100
Bsp119I (BstBI)	50-100
Bsp120I (PspOMI)	20-50	50-100
Bsp1407I (BsrGI)	20-50	50-100
Bsp143I (Sau3AI)	50-100
Bsp68I (NruI)	0	50-100
BspLI (NlaIV)	50-100
BspOI (BmtI)	0	20-50	50-100
BspPI (AlwI)	0	50-100
BspTI (AflII)	0	0-20	50-100
Bst1107I (BstZ17I)	0-20	50-100
BstXI	0		50-100
Bsu15I (ClaI)	50-100
BsuRI (HaeIII)	0-20	20-50	50-100
BveI (BspMI)	0-20		50-100
CaiI (AlwNI)	0	0-20	50-100
Cfr10I (BsrFI)	20-50	50-100
Cfr13I (Sau96I)	50-100
Cfr42I (SacII)	50-100
Cfr9I (XmaI)	20-50	50-100
CfrI (EaeI)	0	50-100
CpoI (RsrII)	50-100
CseI (HgaI)	50-100
Csp6I (CviQI)	50-100
DpnI	50-100
DraI	0	0-20	50-100
Eam1104I (EarI)	0	50-100
Eam1105I (AhdI)	0	50-100
Ecl136II (EcoICRI)	50-100
Eco105I (SnaBI)	20-50	50-100
Eco130I (StyI)	0		50-100
Eco147I (StuI)	0	50-100
Eco24I (BanII)	50-100
Eco31I (BsaI)	20-50		50-100
Eco32I (EcoRV)	20-50	50-100
Eco47I (AvaII)	50-100
Eco47III (AfeI)	0	0-20	50-100
Eco52I (EagI)	0-20	50-100
Eco57I (AcuI)	50-100
Eco57MI	50-100
Eco72I (PmlI)	0-20	50-100
Eco81I (Bsu36I)	50-100
Eco88I (AvaI)	50-100
Eco91I (BstEII)	20-50	50-100
EcoO109I (DraII)	50-100
EcoRI	50-100
EcoRII	0	0-20	20-50	50-100
EheI (SfoI)	20-50		50-100
Esp3I (BsmBI)	50-100
FaqI (BsmFI)	0-20	50-100
FspAI	0-20	20-50	50-100
FspBI (BfaI)	20-50	50-100
GsuI (BpmI)	50-100
HhaI	50-100
Hin1I (BsaHI)	50-100
Hin1II (NlaIII)	0	0-20	50-100
Hin6I (HinP1I)	0		0-20	50-100
HincII (HindII)	50-100
HindIII	0	0-20	50-100
HinfI	50-100
HpaII	0-20	50-100
HphI	0	50-100
Hpy8I (MjaIV)	0-20	50-100
HpyF10VI (MwoI)	50-100
HpyF3I (DdeI)	20-50	50-100
I-SceI	50-100
Kpn2I (BspEI)	0	50-100
KpnI	50-100
KspAI (HpaI)	20-50	50-100
LguI (SapI)	50-100
Lsp1109I (BbvI)	0-20	50-100
LweI (SfaNI)	20-50	50-100
MauBI	0	0-20		20-50
MbiI (BsrBI)	0	0-20	50-100
MboI	50-100
MboII	50-100
MlsI (MscI)	0-20	50-100
MluI	20-50		50-100
MnlI	0	50-100
Mph1103I (NsiI)	20-50	50-100
MreI (Sse232I)	0	20-50	50-100
MspI (HpaII)	0-20	50-100
MssI (PmeI)	20-50	50-100
MunI (MfeI)	20-50	50-100
Mva1269I (BsmI)	0	50-100
MvaI (BstNI)	0	20-50	50-100
NcoI	0	50-100
NdeI	0-20	20-50	50-100
NheI	0	20-50		50-100
NmuCI (Tsp45I)	20-50	50-100
NotI	20-50	50-100
NsbI (FspI)	0	0-20	50-100
OliI (AleI)	0-20	20-50	50-100
PacI	20-50	50-100
PaeI (SphI)	0	0-20		20-50	50-100
PagI (BspHI)	20-50	50-100
PasI	50-100
PauI (BssHII)	0	50-100
PdiI (NaeI)	0-20	50-100
PdmI (XmnI)	50-100
PfeI (TfiI)	50-100
Pfl23II (BsiWI)	0-20	20-50	50-100
PfoI	0	20-50	50-100
Ppu21I (BsaAI)	50-100
PscI (PciI)	0	50-100
Psp1406I (AclI)	0-20	50-100
Psp5II (PpuMI)	0	50-100
PstI	0-20	50-100
PsuI (BstYI)	0-20	50-100
PsyI (Tth111I)	0	50-100
PvuI	20-50		50-100
PvuII	50-100
RsaI	50-100
RseI (MslI)	0	0-20	20-50	50-100
SacI	50-100
SalI	20-50		50-100
SatI (Fnu4HI)	0	50-100
ScaI	0-20	50-100
SchI (MlyI)	50-100
SdaI (SbfI)	0-20	50-100
SduI (Bsp1286I)	50-100
SfaAI (AsiSI)	0	0-20		20-50
SfiI	50-100
SgrDI	0-20	20-50	50-100
SgsI (AscI)	50-100
SmaI	50-100
SmiI (SwaI)	0-20	50-100
SmoI (SmlI)	0	50-100
SmuI (FauI)	50-100
SsiI (AciI)	50-100
SspDI (KasI)	20-50		50-100
SspI	0-20	50-100
TaaI (HpyCH4III)	20-50	50-100
TaiI (MaeII)	50-100
TaqI	0	20-50		50-100
TasI (Tsp509I)	0	20-50	50-100
TatI	0-20		20-50	50-100
TauI	20-50	50-100
Tru1I (MseI)	0	0-20		50-100
TscAI (TspRI)	0	50-100
TsoI	20-50	50-100
Van91I (PflMI)	0	50-100
VspI (AseI)	50-100
XagI (EcoNI)	20-50	50-100
XapI (ApoI)	20-50		50-100
XbaI	20-50	50-100
XceI (NspI)	0	50-100
XhoI	0-20	50-100
XmaJI (AvrII)	50-100
XmiI (AccI)	50-100