An efficient cloning of DNA fragments by a method based on uracil-DNA glycosylase and endonuclease IV.
We introduced a novel method to clone random DNA fragments independent of ligation reaction. The method involves the generation of long protruding ends on PCR amplification DNA. Both oligonucleotides used for the amplification of the vector DNA carried one uracil residue at the tenth position from the 5' end and this made the creation of the 3' protruding ends of linearized vector possible by uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). 76 groups of annealed oligonucleotides that had ten-nucleotides protruding at 3'-end, which were complementary to those at 3'-end of the linearized vector, were designed. The linearized vector and the annealed oligonucleotide were mixed together to transform E.coli directly without ligation reaction. The number of the clone that grew on the plates had been demonstrated to reach 1x10(5) transformants/microg and 96.1% of transformants harbored the cloned fragments. From the results of transformation, we can confirm that the efficiency of the creation of 3' protruding ends in our method is high and our cloning method is benefit to produce recombinants easily and efficiently.
Hou
Jingli
J
College of Life Science & Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Liu
Xipeng
X
Zheng
Yan
Y
Liu
Jianhua
J
D016993:Chlamydophila pneumoniae / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D014162:Transfection; D051981:Uracil-DNA Glycosidase / Q000378:metabolism*
D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D051981:Uracil-DNA Glycosidase