Cleavage Efficiency Close to the Termini of PCR Fragments

When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE, it is very important to determine how many extra nucleotides should be added to the 5’-end of the PCR primer next to the introduced recognition site to ensure efficient cleavage with the appropriate restriction endonuclease. Some restriction endonucleases cleave DNA poorly when their recognition sites are located at the ends of DNA fragments. Information on restriction endonuclease performance close to the end of DNA fragments may be helpful when planning the introduction of cleavage sites at DNA termini in PCR experiments.

Experiments were performed as follows: PCR primers having 1-5 extra nucleotides at the 5’-end of the PCR primers adjacent to the introduced recognition site were 5’-end labeled with [³²P] by T4Polynucleotide Kinase and used in the PCR reaction. Purification of PCR fragment was performed using the modified glass beads procedure as indicated in the DNA Extraction Kit (#K0513) followed by ethanol precipitation. DNA (0.5µg) and 10u of restriction endonuclease were incubated for 1 hour at the recommended incubation temperature and optimal buffer for each enzyme in a total volume of 40µl. Reaction products were separated by 10% PAGE and percent cleavage was determined using OptiQuant Image Analysis Software.

	Cleavage efficiency:
	- 0%
	- 0-20%
	- 20-50%
	- 50-100%
*	- incubation was performed for 16 hours
nd	- not determined

Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
AarI	20-50		50-100
AatII	0	0-20	20-50	50-100
Acc65I	0	20-50
AdeI	50-100
AluI	0	20-50
Alw21I	50-100
Alw26I	50-100
Alw44I	0	20-50	50-100
BamHI	50-100
BcnI	20-50		50-100
BclI	0	20-50	nd
BcuI	50-100
BfiI	50-100
BfmI	50-100
BglI	20-50	50-100
BglII	0	50-100
Bme1390I	20-50		50-100
BpiI	50-100
Bpu10I	20-50	50-100
Bpu1102I	50-100
BseDI	0	50-100
BseGI	50-100
BseLI	0	50-100
BseMI	0-20	50-100
BseMII	50-100
BseNI	0	50-100
BseSI	0	0-20	nd
BseXI	20-50	50-100
Bsh1236I	50-100
Bsh1285I	0-20	50-100
BshNI	50-100
BshTI	20-50	50-100
Bsp68I	0	50-100
Bsp119I	50-100
Bsp120I	20-50	50-100
Bsp143I	50-100
Bsp143II	0	50-100
Bsp1407I	20-50	50-100
BspLI	50-100
BspPI	0	50-100
BspTI	0	0-20	50-100
Bst1107I	0-20	50-100
BstXI	0		50-100
Bsu15I	50-100
BsuRI	0-20	20-50	50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
CaiI	0	0-20
CfrI	0	50-100
Cfr9I	20-50	50-100
Cfr10I	20-50	50-100
Cfr13I	50-100
Cfr42I	50-100
CpoI	50-100
Csp6I	50-100
DraI	0	0-20	50-100
Eam1104I	0	50-100
Eam1105I	0	50-100
Ecl136II	50-100
Eco24I	50-100
Eco31I	20-50		50-100
Eco32I	20-50	50-100
Eco47I	50-100
Eco47III	0	0-20	50-100
Eco52I	0-20	50-100
Eco57I	50-100
Eco57MI	50-100
Eco72I	0-20	50-100
Eco81I	50-100
Eco88I	50-100
Eco91I	20-50	50-100
Eco105I	20-50	50-100
Eco130I	0		50-100
Eco147I	0	50-100
EcoO109I	50-100
EcoRI	50-100
EheI	20-50		50-100
Esp3I	50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
FspAI	0-20	20-50	50-100
GsuI	50-100
Hin1I	50-100
Hin6I	0		0-20	50-100
HincII	50-100
HindIII	0	0-20	50-100
HinfI	50-100
HpaII	0-20	50-100
HphI	0	50-100
Hpy8I	0-20	50-100
KpnI	0	20-50
Kpn2I	0	50-100
KspAI	20-50	50-100
LweI	20-50	50-100
MbiI	0	0-20	50-100
MboI	50-100
MboII	0	20-50		nd
MlsI	0-20	50-100
MluI	20-50		50-100
MnlI	0	50-100
Mph1103I	0-20			nd
MspI	0-20	50-100
MssI	20-50	50-100
MunI	20-50	50-100
MvaI	0	20-50	50-100
Mva1269I	0	50-100
NcoI	0	50-100
NdeI*	0-20		20-50
NheI	0	20-50		50-100
NmuCI	20-50	50-100
NotI	20-50	50-100
NsbI	0	0-20	50-100
Enzyme	Base pairs from site to fragment end
	1	2	3	4	5
OliI	0-20	20-50	50-100
PaeI	0	0-20		20-50	50-100
PagI	20-50	50-100
PauI	0	50-100
PdiI	0-20	50-100
PdmI	0-20	20-50		nd
Pfl23II	0	20-50		nd
Psp5II	0	50-100
Psp1406I	0-20	50-100
PstI	0-20	50-100
PsuI	0-20	20-50	nd
PsyI	0	50-100
PvuI	20-50		50-100
PvuII	50-100
SacI	50-100
SalI	20-50		50-100
SatI	0	50-100
ScaI	0-20	50-100
SchI	50-100
SdaI	0	0-20		20-50
SduI	50-100
SmaI	50-100
SmiI	0-20	50-100
SspI	0-20	50-100
TaaI	20-50	50-100
TaiI	50-100
TaqI	0	20-50		50-100
TasI	0	20-50	50-100
TatI	0-20		20-50	50-100
TauI	20-50	50-100
Tru1I	0	0-20		50-100
Van91I	0	50-100
VspI	50-100
XagI	20-50	50-100
XapI	20-50		50-100
XbaI	20-50	50-100
XceI	0		20-50	nd
XhoI	0-20	50-100
XmaJI	50-100
XmiI	50-100