A SIMPLE,FAST AND EFFICIENT SITE-DIRECTED MUTAGENESIS METHOD

Pure plasmid containing cDNA of murine complement receptor type I gene (mCR2) was mini-prepared. Two pairs of mutagenic primers were synthesized in vitro and the desired two mutations, P15S and T68Y,were introduced into mCR2 cDNA by PCR. After that,DpnI was used to digest the PCR products to remove the methylated,nonmutat-ed parental DNA template. Optimal amount of the digested products were used to transform competent XLl-Blue cells and colonies resistant to ampicillin were picked up randomly followed by being sequenced in order to screen and identify the needed mutant strains. The results show that this novel site -directed mutagenesis method is not only fast and easy to perform,but also very efficient,for the mutagenesis rate is almost