A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study
Jae-Kyun Ko and
Jianjie Ma
Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey
Submitted 25 October 2004
; accepted in final form 17 January 2005
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ABSTRACT
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PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.
Type IIs restriction enzyme; enhanced green fluorescent protein; Bcl-2
PCR-BASED IN VITRO MUTAGENESIS is an invaluable tool for studying protein structure and function in molecular biology and protein engineering (5). Major strategies of PCR-based mutagenesis include base substitution, deletion, insertion, chimeric gene generation, multiple-site mutagenesis, and random mutagenesis at either a single site or multiple sites. Numerous PCR-based methods have been developed commercially or noncommercially. Among those methods, the overlap extension method (2), megaprimer method (3), Quick Change Method (Stratagene, La Jolla, CA), and their modified versions (4, 8, 13) are currently prevalent. However, none of these methods can be applied to all of the diverse mutagenesis strategies mentioned above. As a result, acquisition of a new protocol for various mutation strategies is inevitably required, which is a cumbersome task to researchers.
Herein we report a novel method of generating gene mutations using digestion of PCR-amplified mutagenic products with type IIs restriction enzymes. This versatile mutagenesis technique is quick, efficient, and cost-effective and is of interest to many investigators in cell physiology research. We have demonstrated the fidelity of this method using multiple mutagenesis strategies with enhanced green fluorescent protein (EGFP) and Bcl-2 family cDNAs as templates. Our results reveal that this method was both rapid (within 5 h for generation of a mutated gene) and highly efficient (up to 100% mutation efficiency) in all experiments.
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METHODS
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This PCR-based mutagenesis method takes advantage of the unique features of type IIs restriction enzymes (see Table 1). Briefly, a gene of interest is amplified into two separate PCR fragments using four designed primers. Each fragment is produced by pairing one anchor primer with one mutagenic primer. The two anchor primers contain restriction sites for subcloning of a mutated gene into a vector, and the two mutagenic primers contain a desired mutation near the recognition site of a type IIs restriction enzyme. After digestion of the two PCR fragments with the appropriate type IIs restriction enzyme, cohesive ends of each fragment remain complementary and undergo specific ligation between each other, resulting in mutation. Although similar approaches have been described previously (6, 9), they are limited to mutation of only a single base or a single codon.
The application of this unique method for generation of single-point mutations is shown in Fig. 1A. The 830-bp EGFP cDNA sequence from pEGFP-C1 (Clontech) was amplified into two separate fragments. PCR reactions were performed with 100 ng of template, 1 µM primer pair, 200 µM dNTP, and 2.5 U of Taq polymerase (Takara) in a 50-µl reaction volume. The PCR conditions were 94°C for 4 min for 1 cycle; 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min for 30 cycles; and finally, 1 cycle at 72°C for 7 min. Each reaction was performed with either a primer pair that included a forward anchor primer and a reverse mutagenic primer or a pair that included a reverse anchor primer and a forward mutagenic primer. The sequences of mutagenic primers used in this study are shown in Table 2.

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Fig. 1. A: schematic diagram of the single-codon mutagenesis (G67I) in enhanced green fluorescent protein (EGFP) using EarI type IIs restriction enzyme. Two separate PCR fragments are produced from a template EGFP cDNA. Two mutagenic primers contain mutated codon sequences that are complementary to each other and adjacent to the EarI recognition site (5'-CTCTTC). Each anchor primer contains a restriction site for downstream subcloning. After EarI digestion, two fragments are ligated together to generate the mutated full-length EGFP cDNA. The ligation product is subcloned into the cloning vector between the NheI and XbaI restriction sites. B: electrophoretic analysis of mutagenesis product on a 2% agarose gel. After EarI digestion, PCR fragments F1 and F2 were subjected to ligation reaction with T4 DNA ligase. Lane 1, ligation control reaction in the absence of T4 DNA ligase; lane 2, ligation reaction with T4 DNA ligase.
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Amplified fragments were digested with 10 U of EarI restriction enzyme at 37°C for 1 h, resulting in exposure of complementary cohesive ends. The digested fragments were purified by standard phenol extraction and then ligated with 400 U of T4 DNA ligase (New England Biolabs) at room temperature for 1 h to generate 830-bp EGFP cDNA containing a desired mutation (Fig. 1B). The ligated fragments were then purified from 2% agarose gel using the QIAquick gel extraction kit (Qiagen) and subcloned into a cloning vector after double-digestion with NheI and XbaI. For each mutation generated, five plasmids containing inserts were isolated and sequenced to confirm the presence of the desired mutation.
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RESULTS
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While various mutagenesis protocols based on the PCR method have been developed, most of them require several rounds of mutagensis to generate multiple mutations and produce relatively low mutagenesis efficiency. In the present study, to introduce three separate mutations in EGFP, three PCR fragments were produced in separate reactions with the primer pairs listed in Table 2 (Cycle3). As shown Fig. 2A, to avoid the practical difficulty with PCR amplification of small cDNA fragments, a synthetic nucleotide sequence (F3) encoding amino acid residues between two adjacent mutations was used in the ligation reactions. After PCR, two ligation steps were performed sequentially. First, fragments F1 and F2 or F3 and F4 were ligated separately to produce intermediate ligation products (Fig. 2B, lanes 2 and 4). Second, these two intermediate ligation products were mixed to allow further ligation. Finally, the end ligation product, represented by the top band in lane 6 of Fig. 2B, was purified from the agarose gel and subcloned into the cloning vector. As shown in Table 3, this mutagenesis strategy produced near-perfect end products of the desired mutations. The mutagenesis efficiency at the desired three sites was estimated at 100% using DNA sequence analysis from five clones, and four correct clones were obtained without additional mutation resulting from PCR fidelity errors.

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Fig. 2. A: schematic diagram of multiple-site mutagenesis at three separate codons. F1, F2, and F4 fragments were generated by performing PCR using the specific primers listed in Table 2. F3 is a 30-bp synthetic oligonucleotide fragment, coding amino acids M153V163 of EGFP. After EarI digestion and ligation reactions, a full-length ligation fragment is generated and then subcloned into a cloning vector using NheI and XbaI cloning sites. B: electrophoretic analysis of mutagenesis product on a 2% agarose gel. Lane 1, ligation control reaction of F1 and F2 without T4 DNA ligase; lane 2, ligation reaction of F1 and F2 with T4 DNA ligase; lane 3, ligation control reaction of F3 and F4 without T4 DNA ligase; lane 4, ligation reaction of F3 and F4 with T4 DNA ligase. The two intermediate ligation reactions were mixed and further incubated on the ice (control reaction, lane 5) or at room temperature (lane 6) for 1 h. Bands a, b, and c indicate intermediate fragments F1+F2, F3+F4, and the full-length fragment F1+F2+F3+F4 of EGFP, respectively.
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DISCUSSION
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The mutagenesis method we describe herein is rapid and highly efficient for introducing specific mutations into any site of a target DNA sequence. This method has several advantages. First, it is simple and cost effective. Second, mutated genes can be generated rapidly. Typically, mutation at a single site or at multiple sites (up to 3 separate positions) can be produced in as little as 5 h during experiments. Third, the PCR-based mutagenesis strategy has very high efficiency and fidelity.
Perhaps the most unique aspect of this strategy is the ability to create random, multiple mutations in a given gene. As shown in Fig. 3, different mutations at a given site can be produced using randomized PCR primers. This strategy is useful for directed protein engineering studies. Moreover, with specific design of the mutagenic primers, this method can be used to create insertions, deletions, and chimeric genes as shown in Fig. 4.

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Fig. 3. Experimental strategy for random mutagenesis at the multiple sites. The overall experimental scheme is similar to that of the multiple-site mutagenesis described in Fig. 2A, except for the design of mutagenic primers, which contain randomized codon sequences near the cleavage site of the type IIs restriction enzyme. Additional codons can be mutated by performing repeated mutagenesis reactions using the first-round mutated gene product as a PCR template and additional random mutagenic primer sets. The codons shown were chosen arbitrarily for illustrative purposes.
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Fig. 4. Various mutagenesis strategies using type IIs restriction enzymes, including insertion (A), deletion (B), and chimeric gene generation (C). Codons shown were chosen arbitrarily for illustrative purposes.
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Although we used EarI restriction enzyme for our study with EGFP, other type IIs restriction enzymes, such as SapI and BsmBI (Table 1), also can be useful. For example, we used SapI restriction enzymes for our mutagenesis of Bcl-2 family genes as shown in Tables 2 and 3. Similarly to EarI, highly efficient mutagenesis results with Bax and Bcl-xL were achieved with SapI. The rapid and efficient generation of multiple mutations will allow investigators to examine the structural and functional relationship of a given gene in future studies.
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GRANTS
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This work was supported by National Institutes of Health Grants R01 AG-15556, R01 CA-95739, R01 HL-69000, and R01 DK-51770.
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ACKNOWLEDGMENTS
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We thank Dr. Noah Weisleder for helpful advice in manuscript preparation.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. Ma, Dept. of Physiology and Biophysics, Robert Wood Johnson Medical School, Univ. of Medicine and Dentistry of New Jersey, 675 Hoes Lane, 5th Floor, Research Tower, Piscataway, NJ 08854-5635 (E-mail: maj2{at}umdnj.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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