Abstract: Simplifying overlap extension PCR（OE-PCR）protocol in site-directed mutagenesis by parallel template is not valid. The low efficiency of restriction enzyme cleavage close to the end of DNA fragments is the key reason of restricting the site-directed mutagenesis by OE-PCR. The enzymatic digestion efficiency of DNA product is improved by Outboard-primers method,and as a result,the achievement ratio of site-directed mutagenesis increases. The matching loci in upstream and downstream primer were outward moved by 50-100 bp with a pair of outboard-primers;therefore,the enzymatic digestion sites at both ends of target gene were far from the ends of the second round PCR product. Thus,the subsequent enzyme digestion efficiency was improved and the reaction time was saved. Because the length of DNA was obviously shortened by enzymatic digestion at this time,the results of gel electrophoresis showed that the PCR product was fully digested in only 1 h. The number of clone after ligating the transformation of the products into receptive Escherichia coli also increased significantly,and the positive rate increased from 33%-70% to 100%. Comparing with traditional terminal primers,outboard-primers method not only saves time,but also remarkably increases the positive colony number and success rate in site-directed mutagenesis of overlap extension PCR.

Key words: overlap extension PCR, site-directed mutagenesis, outboard-primer