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Restriction Endonucleases: Cleavage Efficiency Close to the termini of PCR Fragments
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Digestion of PCR Products

 

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 T4 Polynucleotide 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