OL-PCR for site-directed mutagenesis of full-length cDNA clone of DEN-2
Objective To generate the oligonucleotide directed mutants of the full length cDNA clone of dengue 2 virus. Methods Two DNA fragments with single point mutation (E62 or E203) were amplified with four pairs of oligonucleotide primers by OL PCR and then cloned into pGEM T vectors respectively. The recombinant T vectors, T TB62 and T TB203, were digested with ClaⅠ+SphⅠand SphⅠ+NheⅠ, then ligated to pDVWS501 with T4 DNA ligase respectively. The recombinant plasmids, TB62 and TB203, were sequenced. Results The results of DNA sequencing indicated that TB62 and TB203 with point mutation were attained. Conclusion OL PCR is a kind of site directed mutagenesis method to achieve high mutation efficiency. [