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

1. Background
    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 site-directed 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 the gene specific primers). These two additional primers are dependent  on the recombinant vector that harbors the gene (i.e. M13/PUC or T7 promoter universal primers). These primers are designed such that to position at least 50 to70 base pairs outward from binding site of the nested primers C and D.
    The proposed procedure  consists of four steps as follows: (i) The target gene is amplified by PCR using gene specific primers (nested primers C and D) and is cloned into the vector (i.e. pTZ57R/T or pGEM).
(ii) The target gene in recombinant vector (0.1-0.2 ng) will be subjected to the first round of PCR using A and F primers and the second PCR using E and B primers to amplify AF and EB fragments, respectively (FigUre 1).
(iii) The amplified fragments are purified and used as template to perform the third round of PCR by N-SOE method (Figure 1).
(iv) Equimolar amounts of the first and second PCR products (about 0.2 ng) and  10 pmol of each C and D primers (nested primers) are added to the PCR reaction for overlap extension and amplification of the full length of the mutated gene (Figure 1). The first 10 cycles of the third PCR are performed with shorter extension time at 68ºC, followed with 25 cycles with longer extension time at 72°C. The full size of the mutagenized fragment  will be purified from agarose gel and will be cloned into the cloning vector respectively.
    To validate the specificity of the proposed N-SOE method, an experiment was designed in order to introduce a specific mutation in the Bacillus thermocatenulatus lipase gene (btl2) and compare it with the conventional SOE.

2. Objectives
    The aim of the present  study is to improve SOE-PCR that is called N-SOE-PCR to enhance the specificity of  mutagenesis.

3. Materials and Methods

3.1. Cloning of the btl2 Gene
    Bacillus thermocatenulatus genomic DNA was extracted according to the method described by Sambrook et al. (2001). All the PCR reactions were performed in a final volume of 50 mL containing 200 mM of each dNTP, 5 mL of PCR buffer with a final concentration of 1.5 mM MgCl2, 5% glycerol, 2 units Pfu DNA polymerase, 10 pmol of each primer and 2 ng Bacillus thermocatenulatus genomic DNA  as template.  The lipase gene (btl2) was amplified by using the gene specific primers (primer C; 5¢-GATGGCCATGGCGGCATCCCCACGCGCC-3¢, Mlu NI site and primer D; 5¢-TTGAGCTCATCATCCCTTCATTAAGGCCGC-3¢, SacI site) designed according to btl2 gene sequence (GenBank X95309). The PCR reaction was carried out in a thermocycler (Techne, UK) with the following settings: 4 min at 94°C, 1 min at 94°C, 1 min at 62°C, and 1.5 min at 72°C) for 30 cycles with a final cycle of 5 min at 72°C. Subsequently, the amplified gene (~1200 bp) was cloned into pTZ57R/T vector leading to the formation of the pTA-BTL2 plasmid, which was introduced into E. coli DH5a.

3.2. SOE and N-SOE-PCR Method
    The standard SOE and N-SOE-PCR methods were used in order to generate mutation in the btl2 gene. 2 ng of the recombinant pTZ-BTL2 plasmid was used as template in the first and second PCR reactions. In both methods, the first PCRs were performed by using the primer pairs of A and F (M13/pUC forward primer; 5¢-TAAAACGACGGCCAGTGAATTCG-3¢ and the reverse mutated primer; 5¢-CTGATCGCGCCTTTGACCTGC-3¢. The mutated nucleotides are shown in bold respectively. The second PCR was performed by using the primer pairs of E and B such that the forward mutated primer was; 5¢-GCTGGTCAAAGGCGCGATCAG-3¢ and the M13/pUC reverse primer was composed of 5¢-GAAACAGCTATGACCATGATTACG-3¢). The first and second PCRs were performed under following the conditions: 2.5 min at 94°C, (1 min at 94°C, 1 min at 48°C, and 45 sec at 72°C) for 30 cycles and a final step of 5 min at 72°C.  These two PCR fragments were purified using High Pure PCR Product Purification Kit (Roche, Germany) and served as a template for the subsequent third round of PCR. In the N-SOE  method the third PCR is performed, whereas in the standard SOE method the third PCR is performed by addition of primer pairs of A and B to the PCR master mix just after 10 cycles of the third PCR amplification (Figure 1). In both methods, the third PCR was carried out using the following settings: 1.5 min at 94°C, (45 sec at 94°C, 1.5 min at 48°C, and 45 sec at 68°C) for 10 cycles, and (45 sec at 94°C, 1 min at 56°C, and 1.5 min at 72°C) for 25 cycles with a final cycle of 5 min at 72°C. Subsequently, PCR product yield and quality was analyzed by agarose gel electrophoresis. All the PCR reactions were repeated four times.

4. Results
    In the first and the second PCR reactions a ~600 bp (Figure 2A; lane a,) and a ~740 bp (Figure 2A; lane b) fragments are amplified, respectively. These two PCR products were served as template for the subsequent third PCR amplification. In the N-SOE method the third PCR is performed by using C and D primers to obtain a ~1200 bp fragment (Figure 2B; lanes G-J), whereas in standard SOE method the third PCR is performed with the A and B primers to obtain a ~1300 bp fragment (Figure 2B; lanes G-J). The third PCR products of the N-SOE and the standard SOE method were cloned in pTZ57R/T to produce recombinant pTA-M-BTL2 plasmids. Subsequently, the presence of the mutation into the btl2 gene was confirmed by DNA sequencing (Data not shown). In N-SOE method, the product of the third PCR (~1200 bp) was slightly smaller than standard method (~1300 bp). The reason was that  gene specific primers were positioned in the interior part of the fragment, at least 50-70 bp apart far from it’s both ends.

5. Discussion
    Site-directed mutagenesis is a method that is used to make changes to the any gene. Until now, many techniques have been developed to improve the efficiency of mutagenesis. In this study the standard SOE  method improved to increase the efficiency of mutagenesis of any gene. The expected fragments in the SOE and N-SOE methods were approximately 1300 and 1200  bp respectively. As we expected, using the N-SOE method a single fragment of the correct size (~1200 bp) was amplified without any detectable background, whereas applying SOE method in addition to the expected fragment (~1300), an extra fragment (~600 bp) was also amplified. (Figure 2, lanes G to J, 600 bp fragment).
    In the N-SOE method, similar to the conventional PCR, the specific or nested primers are added to the PCR master mix just in the first cycle of the third PCR,  whereas in a standard SOE method primers are added to the PCR master mix after 10 cycles of the third PCR.
    To summarize, in the N-SOE method (i) using a set of additional primer, (ii) addition of gene specific primers (C and D primers) to the PCR master mix just at the first cycle of third PCR and (iii) using extension temperature of 68°C instead of 72°C in first 10 cycles of the third PCR will result in an increased specificity of SOE method. These alterations in SOE make the method more productive and efficient in the experiments wherein site-directed mutagenesis will be subject of application.

Acknowledgments
We would like to thank H. Ahmadi Danesh for the valuable technical helps.