Efficient Multi-site-directed Mutagenesis directly from Genomic Template. Fengtao Luo 1, Xiaolan Du 1, Tujun Weng 1, Xuan Wen 1, Junlan Huang 1, Lin Chen 1 Running title: Multi-site-directed Mutagenesis from Genomic Template. 1. State Key Laboratory of Trauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042 China Corresponding author. Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042 China. Tel: +86-023-68757041; Fax: +86 2368702991; Email address: linchen70@tmmu.edu.cn (L. Chen) Abstract: In this paper, traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well as for combination, deletion, or insertion of DNA fragments. Keywords: Multi-site-directed mutagenesis; Overlap extension; PCR; 3 -UTR; Genomic template 1. Introduction As a fundamental technique, site-directed mutagenesis is a powerful tool in investigating the function of regulatory DNA sequence such as promoter, intron, 5 - or 3 -untranslated region (UTR) (Higuchi et al 1988; Ito and Lai 1997). Generally, the desired change is only with one site, but sometimes one need simultaneously to introduce multiple mutations in different positions in a gene to understand the DNA function or to optimize the gene expression. Among a variety of multi-sitedirected mutagenesis (MSM) approaches, overlap extension PCR (OE-PCR) and quick-change multi- 1
site-directed mutagenesis system developed by Stratagene Company are predominately used owing to their simplicity and efficiency. Quick change method is simple for MSM, but it requires circular plasmid as amplification template (Wang and Malcolm 1999; Hogrefe et al 2002). To use this method, non-mutated target sequence coming from genomic DNA or cdna must be firstly cloned into plasmid, and then the plasmid is employed for MSM. Thus, Quick change method is only simple for plasmidbased mutagenesis. OE-PCR is traditional approaches widely used for site-direct mutagenesis (Ling and Robinson 1997). Compared to Quick change method, OE-PCR is advantageous for MSM with respect to linear template. In this method, firstly, two or more mutation-containing fragments are amplified separately by using one universal and one mutagenic primer or two mutagenic primer pair. The two or more intermediate products with complementary ends form a new template DNA by allowing the 3 overlap of each strand to serve as primer for the 3 extension of the complementary strand. Then the mutant DNA is generated from the fused new template DNA through PCR amplification using two universal primers (Ling and Robinson 1997). In traditional OE-PCR approach, it s necessary to remove wild-type DNA template and remaining primers in the first amplification, so purifying the products of the first PCR reaction by gel electrophoresis is needed prior to overlap extension (Peng et al 2006). This purification step is laborious and cost consumptive. Although improved megaprimer-pcr method and asymmetric overlap extension PCR have been reported bypass intermediate product purification step, these methods are preferable for single site mutagenesis (Tyagi et al 2004; Xiao et al 2007). Further more, most of reported MEM strategies based on OE-PCR employed plasmid as test template, and under the condition of genomic template, we confronted many difficulties when using these methods. Thus, it is still necessary to improve the OE-PCR method for MEM and simultaneously to simplify its procedure suitable for genomic template. In this study, we present an efficient OE-PCR method for MSM directly from genomic template with simplified procedure. 2. Materials and Methods As depicted in Fig 1A, for two-site mutagenesis, the template DNA were divided into three 2
fragments (I, II and III) by 2 desired mutation sites, and three pairs of primers(f1, R1, F2, R2, F3 and R3) for amplifying these fragments were used. There were two direction-converse primers at each mutagenic site including complementary region (15-20bp) at their terminal ends to splice overlap extension. The mutagenic site was located in the center of two converse primers or at the 5 end of one of the two primers. To avoid amplification of non-mutated template genomic sequence in full-length PCR reaction, two flanking sequences, containing suitable restriction enzyme cutting sites, were added at the 5 ends of primer F1 and R3 respectively, which providing combination sites for full-length forward (FF) primer and full-length reverse (FR) primer respectively. Low concentration of primers was used so that the primers could be used up after fragment amplification, which provides no remaining primers in subsequent overlap extension and full-length fragment amplification. To describe the improved OE-PCR mutagenesis method in detail, we used the 3 -UTR region of mouse fibroblast growth factor receptor 1 gene (Fgfr1, gene ID: 14182) gene as test template. Online software prediction demonstrated that there are two potential microrna-binding sites at 261-267bp and 491-496bp positions of mouse Fgfr1 gene 3 -UTR. We plan to mutate two positions located at 263-264bp and 493-494bp of Fgfr1 gene 3 -UTR sequence using our improved OE-PCR mutagenesis method described above. Primers designed to amplify fragments (I, II, and III) or full-length sequence and their positions are shown in Table 1. The desired mutation is contained within the overlapping primer sequences and three step PCR reactions were adopted. Firstly, three fragments were amplified separately in a 30 µl volume with 200 ng mouse genomic template, 1.25U primerstar polymerase (Takara), 6µL 5 primerstar reaction buffer, 160µM dntps, and 2µL specific concentration (0.5µM) of primer pair mixed with 1:1 ratio. Primers F1 and R1 were employed to amplify fragment I (about 260bp), similarly, F2 with R2 were used to amplify fragment II (about 250bp) and F3 with R3 for obtaining fragment III (about 600bp). The PCR conditions were initial denaturizing at 95 for 5 min, followed by 20 cycles of denaturizing at 95 for 20 s; annealing at 55 for 30 s; extension at 72 for 45 s, and subsequently incubated at 72 for 5 min. Secondly, 10µL PCR product of each fragment amplification were taken out and a mixture were prepared by mixing them together in a new tube. Then the mixture was subjected to overlap extension, under the following conditions: 5 cycles of 95 for 20 s; 45 for 30 s; 72 for 1 min 20 s, 3
incubated at 72 for 5 min. Thirdly, after adding 1µL outmost primers FF and RR (10µM), containing Mlu I and Xho I site respectively, into the overlap extension product, full-length mutated sequence (1100bp) of Fgfr1 3 -UTR were obtained by routine PCR reaction with amplification conditions as: 95 for 5 min, 30 cycles of 95 for 20 s; 55 for 30 s; 72 for 1 min 20 s, and 72 for 5 min. Then, 3µL of the reaction product was subjected to agarose gel electrophoresis to detect interested band. 3. Results After three step PCR reactions, from agarose gel electrophoresis image, we observed brilliant desired band located at position about 1100 bp (Fig 1B. lane 5), indicative of successfully amplified sequence containing mutation. To illustrate the effect of different primer content on the amplification product, we set a series concentration of primers for fragment amplification, which ranging from 10µM to 0.05µM (Fig 1B. lane 2-7). We found, from high to low of primer concentrations, the interested band can be obtained regularly. When high concentration of primers (10µM) was adopted, besides several nonspecific products, no visible band existed in 1100bp position (Fig 1B. lane 2). Interestingly, when primer concentration was lowered to 5µM, faint band of interest was detected but obvious nonspecific amplification products also were observed (Fig 1B. lane 3). Subsequently, using 1µM or 0.5µM concentration of primers, abundant desired amplification products were detected without obvious nonspecific band (Fig 1B. lane 4 and 5). These results demonstrated that, in the fragment amplifications, lower concentration of primers (1µM-0.5µM) was suitable for obtaining desired mutated product, probably because absence of remaining primers could not disturb the following overlap extension and final full-length fragment amplification. However, if primers used were too little (<0.1µM), only some faint bands were detected owing to the lack of primers to obtain enough PCR product. The results suggest that primer concentrations ranging from 1µM to 0.5µM is appropriate for this improved method. To explore the possibility of further simplifying the procedure, we amplified fragments I, II and III in the first step of PCR reaction in one tube by using just 1µL of each primer at a concentration of 0.5µM, however, the final full-length fragment was faint, and was difficult to be retrieved. Fig 1B (lane 1) shows the result from one experiment, in which the first 10 cycles-pcr was carried out to amplify 4
fragment I and III, and subsequently 10 cycles to amplify fragment II in one tube. We can notice that there is a weak PCR product at 1100 bp position with some bands of non-specifically amplified products. After observing the interested band in electrophoresis, the correct PCR product was purified by gel extraction using the Takara agarose gel DNA purification kit. In order to remove flanking sequence created by primers F1 and R3 and to facilitate insertion into vector, the purified products were digested with 5U MluI and XhoI. Then the MluI/XhoI digested fragment was cloned into the same enzyme digested pgl3-m plasmid using solution I ligase. The combined products were transformed to Escherichia coli (DH5α) and approximately 50 ampicillin resistant single colonies were formed in the plate after incubation at 37 overnight. A total of 10 plasmids of single colonies, randomly selected from plate, were extracted and identified with MluI/XhoI restriction enzyme digestion. Then 7 recombinant clones were subjected to DNA sequencing. Results showed all of the recombinant clones containing 2 expected mutations. Gel electrophoresis image and DNA sequencing results demonstrated that, using the improved OE-PCR mutagenesis method described above, we successfully mutated four nucleotides of two positions located in 3 -UTR of Fgfr1 gene (263-264bp and 493-494bp) directly from eukaryotic genomic template (Figure 2). 4. Discussion As the complication of genomic sequence, which easily resulting in mis-priming in PCR amplification, it was difficult when using common OE-PCR for MSM from it. To date, rare papers reported using genomic template to generate MSM. The POEP method (polyacrylamide gel electrophoresis-mediated overlap extension polymerase chain reaction) developed by Peng et al was efficient for MSM from bacterial genome, but it needs polyacrylamide gel electrophoresis to purify the intermediate product, which complicated the procedure (Peng et al 2006). Compared to POPE method, our method was simpler. Using plasmid template, An et al simplified traditional OE-PCR for MSM by mixing every two adjacent fragments to avoid purification step (An et al 2005). In our improved OE- PCR method, the procedure is further simplified by adding flanking sequences in the 5 end of F1 and R3 primers, which efficiently ensure that only the correct full-length fragments are amplified and simultaneously guarantee the success of mutagenesis as long as there is rare overlap extension template, 5
making this method simplifies the routine work, and therefore saves the time and experimental cost. The method depicted in this study is efficient for MSM, including very close mutation sites (theoretically only if the distance is longer than primer length). As is known, OE-PCR displays higher efficiency than other site-directed mutagenesis methods. In our method, the efficiency reaches up to 100%. In summary, we have improved traditional OE-PCR method and developed a simple, rapid, and efficient multi-site-directed mutagenesis method. With this method, mutated fragment could be generated directly from genomic DNA or cdna, without requirement for ligation of mutated fragments, or purification of intermediate PCR products. Thus, it will be useful for obtaining mutated sequence from cdna or genomic sequence. Furthermore, this improved OE-PCR method will also be applied for the combination, deletion, or insertion of DNA fragments. Acknowledgments We thank Zhenle Zang for her advice in PCR amplification. This work was supported by the National Natural Science Foundation of China (No. 30971607, and No. 81170809). References An Y, Ji J, Wu W, Lv A, Huang R and Wei Y 2005 A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR; Appl. Microbiol. Biotechnol 68 774-778 Higuchi R, Krummel B and Saiki R 1988 A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions; Nucleic Acids Res 16 7351-7367 Hogrefe H H, Cline J, Youngblood G L and Allen R M 2002 Creating randomized amino acid libraries with the QuikChange multi site-directed mutagenesis kit; Biotechniques 33 1158-1165 Ito T and Lai M 1997 Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome; J. Virol 71 8698-8706 Ling M M and Robinson B H 1997 Approaches to DNA mutagenesis: an overview; Anal. Biochem 254 157-178 Peng R H, Xiong A S and Yao Q H 2006 A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis; Appl. Microbiol. Biotechnol 73 234-240 6
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