Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.
The cDNA of human Interleukin\|18 was obtained by RT\|PCR from the mRNA of human peripheral blood cells.The DNA sequencing was performed and the DNA was cloned into expression vector pBV220.The overexpression was performed in E.coli HB101.The expressive product was purified through Sephadex G100 and identified by N\|terminal amino acid sequencing and Western blot.
