A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously.
Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.
An
Yingfeng
Y
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, PR China.
Lv
Anguo
A
Wu
Wenfang
W
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000737:chemistry; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004274:DNA, Recombinant; C056436:endodeoxyribonuclease SpeI; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases