[An improved PCR-based megaprimer method for site-directed mutagenesis].

摘要: Objective To establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM). Methods This protocol is based on the design of two different plasmid DNA templates. Template 1 lacks binding site reverse flanking primer, template 2 forward primer. modification avoids amplification full-length wild-type sequences while templates, primers exist simultaneously in one reaction tube. A synthesized first using 1, primer mutagenic The that needn't cumbersome gel purification step directly added to second system. reaction(PCR 2) containing stages performed 2, megaprimer, During stage extended form mutation product. In SDM residues subsequently amplified with primers. All final products contained desired mutation. Results Fifteen types rare beta-thalassemia mutations Chinese were obtained this method. Each these modified fragments was separately cloned into pGEM-T vector sequenced. involved mutagenesis amplicons identified all clones. Conclusion improved PCR-based rapid, highly efficient, success rate could reach 100%. Furthermore, suitable routine application molecular cloning.