A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method

Site-directed mutagenesis is widely used in molecular biology either in the studies related to the structure-function of proteins or for engineering of proteins that are useful and enhancement of the enzymes’ activity or their resistance to the environmental conditions as well as chemicals (1, 2). A variety of methods that are based on polymerase chain reaction (PCR), including overlap extension, have been established for sitedirected mutagenesis (3-7). Splicing by overlap extension (SOE) provides a powerful method to generate recombinant sequences without a dependence on the restriction sites or ligases (8, 9). However, the major drawback of SOE is its low specificity in the amplification of the full size mutated fragment in the third PCR. In order to challenge this issue, in the present study we have introduced an improvement into the standard SOE method as we called it N-SOE method. This innovation has improved the specificity of mutagenesis. A schematic presentation of the N-SOE method is depicted in Figure 1. In this method, three pairs of primers, namely A and B, C and D, and E plus F, are used. Primers C and D are gene specific, and primers E and F are the mutated primers in a standard SOE method. Primers A and B are additional complementary primers that flank (i.e. locate to the outside) the standard SOE with respect to the primers C and D (or Iran J Biotech. 2015 June;13(2): e1090 DOI:10.15171/ijb.1090