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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