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Use of Class IIS Restriction Enzymes for Site-Directed Mutagenesis: Variations on Phoenix Mutagenesis
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NOTES & TIPS Use of Class IIS Restriction Enzymes for Site-Directed Mutagenesis: Variations on Phoenix Mutagenesis Toshiro Shigaki and Kendal D. Hirschi 1 USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, Texas 77030 Received April 4, 2001; published online September 28, 2001
This report describes a method for creating a mutation in a plasmid without reference to restriction sites. This method requires the synthesis of two primers and PCR to copy an entire supercoiled plasmid. One of the primers contains the mutated region together with a Class IIS restriction site near its 5⬘ end, whereas the other, which is targeted to the same region of the plasmid, bears a recognition site for the same enzyme, also near the 5⬘ end. Upon cleaving the hybridized PCR products with the chosen restriction enzyme, the recognition sites will be jettisoned, and staggered ends that permit seamless joining of the linear products into circular plasmids are created. Hence the primers contain the only required enzyme recognition sites. However, as the entire plasmid is generated by the polymerase chain reaction, it is necessary to sequence all parts of the product in which mistakes of any type cannot be tolerated. Commercially available kits have not removed all troublesome elements from the process of creating mutations in your favorite gene (YFG). 2 Fortunately, an elegantly straightforward technique, which does not require a kit, was developed to circumvent many of the problems (1, 2). In this report we have added variations to that procedure, which is called “phoenix mutagenesis,” to extend its utility. Phoenix mutagenesis makes use of restriction enzymes called hapaxoterministomers that cleave DNA into fragments bearing staggered ends with virtually unique sequences. Hapaxoterministomers are drawn 1 To whom correspondence should be addressed. Fax: (713) 7987078. E-mail: [email protected]. 2 Abbreviations used: YFG, your favorite gene; OFG, our favorite gene.
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from Class IIS restriction enzymes, which cleave outside the recognition sequence, and from enzymes that cleave within interrupted palindromic sequences. Because the overhangs of every fragment so generated are almost always different, each fragment can be joined only to those fragments with which it was originally contiguous. Thus, a plasmid cut with hapaxoterministomers into several fragments can be reborn (hence the name Phoenix) simply by ligating. In such a system, mutations can be created by swapping a fragment of the original template with an engineered fragment bearing both the desired mutation and the ends identical to those of the fragment it was designed to replace. To substitute one fragment for another, the mutated fragment can be added to an unfractionated mixture of the original fragments where it competes with its unmutated couterpart for a place in the finished, circularized plasmid. If the competition is unsuccessful, the superfluous fragment must be removed from the ligation mixture before the mutant counterpart is added. A major advantage of the method is that only the engineered fragment must be sequenced; all others are unchanged. A drawback is that recognition sites for hapaxoterministic enzymes must flank the site of the desired mutation. Our method dispenses altogether with the need for any restriction sites in the target plasmid. Instead, we introduce specific Class IIS restriction sites into the primers used for PCR amplification. This makes the procedure applicable to any gene, regardless of the sequence. The presence of such sites in the plasmid is irrelevant because the final reconstitution of our plasmid occurs in a manner identical with that of the Phoenix system. We believe the development of this modification of the Phoenix protocol is particularly advantageous for the introduction of multiple amino acid changes into YFG. Figure 1 shows in schematic form an example of our mutagenesis approach. In this example, we were interested in altering nine amino acids (27 nucleotides) of our favorite gene (OFG; Fig. 1a). If we were to use commercially available kits, we might first need to subclone the fragment into the appropriate vector; then, after the mutagenesis we would need to shuttle OFG back into the cloning vector for further manipulations. However, utilizing the Phoenix-based variation, we avoid these complications. Initially, a set of Analytical Biochemistry 298, 118 –120 (2001) doi:10.1006/abio.2001.5341 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
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FIG. 1. An example of site-directed mutagenesis with the Class IIS restriction enzyme BsmBI. (a) The 27 bases of wild-type DNA (upper sequence, underlined) are mutated to the bases indicated by bold letters in the lower sequence. Corresponding amino acid sequences are also shown. (b) Primers used in the mutagenesis. Primer A (the forward primer) consists of the following elements starting at the 5⬘ end. (1) Six random bases for the efficient digestion by BsmBI. (2) BsmBI recognition sequence (boxed). (3) An extra (random) nucleotide as a spacer. This and all other upstream nucleotides are cleaved off by BsmBI. (4) CATT (underlined) will be the overhang created by BsmBI and it will be complementary to the overhang on Primer B. This is not required for annealing as mentioned in the text. (5) Desired mutation sequence as shown in bold letters that is 27 nucleotides long. (6) Twenty-one nucleotides (underlined) that are hybridized to the plasmid template. Primer B (the reverse primer) consists of the following elements starting at the 5⬘ end. (1) Six random bases for efficient digestion. (2) BsmBI recognition sequence (boxed). (3) A random spacer nucleotide. (4) The next four nucleotides, AATG (underlined), both serve as hybridizing sequence and will be a sticky-end overhang when digested with BsmBI. (5) Twenty-one nucleotides (underlined) hybridized to the template. (c) Primers A and B are used to amplify the supercoiled plasmid template to produce an entire, but linearized plasmid. BsmBI sites are shown by a bold line, the introduced mutation sequence is shown by a bold broken line, and the annealing sequences are shown by regular lines parallel to the plasmid template. (d) The double-stranded DNA made by the PCR amplification is shown in detail. Corresponding amino acid sequences are also shown. The linear DNA is digested with BsmBI (the recognition sequence is indicated by a box) to make complementary overhangs at both ends. Gaps have been introduced to show the position of the cleavage sites (small arrows). The digested DNA is then column-purified to remove the small fragments (indicated by italic letters) with a BsmBI recognition sequence. The purified sticky-ended DNA is then simply self-ligated to produce the gene with 27 nucleotide (9 amino acid) long mutations.
primers targeted to the intended mutation site (Fig. 1b) was made in order to amplify the whole plasmid linearly (Fig. 1c). Primer A contains an introduced stretch of nucleotides that are changed to code for the
altered nine amino acids (bold letters, Fig. 1b) upstream of the annealing sequence (underlined, Fig. 1b). At the 5⬘-end of the primer, a BsmBI (a Class IIS enzyme) site was added, along with extra bases (TA-
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NOTES & TIPS TABLE 1
Type IIS Restriction Enzymes That Can Be Used for Site-Directed Mutagenesis Enzyme BbvII BsmAI BspMI Eco31I BsmBI (Esp3I) FokI HgaI SfaNI Sth132I
Recognition sequence and cleavage site 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘
GAAGACNNˆNNNN_ 3⬘ GTCTCNˆNNNN_ 3⬘ ACCTGCNNNNˆNNNN_ 3⬘ GGTCTCNˆNNNN_ 3⬘ CGTCTCNˆNNNN_ 3⬘ GGATGNNNNNNNNNˆNNNN_ 3⬘ GACGCNNNNNˆNNNNN_ 3⬘ GCATCNNNNNˆNNNN_ 3⬘ CCCGNNNNˆNNNN_ 3⬘
Note. The sequence is for only one strand, and the cleavage site on this strand is indicated by ˆ . The cleavage site on the complementary strand is indicated by _.
GATG) to ensure efficient digestion. The cutting site of BsmBI is one base away from the recognition sequence CGTCTC and five bases away from the complementary recognition sequence GCAGAG. This leaves a fourbase cohesive end within the coding region of OFG. In this example, the sequence of these four bases must originate from the native gene. These bases are complementary to the template, but hybridization to the template is not required for successful mutagenesis. This four-base-pair cohesive end is compatible with the other end amplified at Primer B when digested with the same enzyme (Fig. 1d). We performed PCR in a 100-l volume containing 100 ng of supercoiled plasmid template (4.6 kb), 50 pmol each of forward and reverse primers, 20 nmol dNTP, 1.5 mM MgCl 2, and 2.5 U Expand High Fidelity DNA polymerase (Roche, Indianapolis, IN). The thermocycler was programmed at 94°C for 30 s for denaturation, 58°C for 1 min for annealing, and 68°C for 4 min for elongation, and the cycle was repeated 30 times. The PCR products were column-purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and digested with DpnI to destroy all the wild-type DNAs (the templates for PCR amplification). This restriction enzyme recognizes only dam-methylated DNA such as the plasmid template, leaving the PCRamplified DNA intact (3). Following column purification, the DpnI-digested PCR products were digested with BsmBI and columnpurified again to remove the small DNA fragments where the BsmBI site is located in order to prevent these fragments from competing with the legitimate sticky ends for ligation. The digested fragment was then simply self-ligated at 16°C for 16 h to recreate a seamless coding sequence, but with a large introduced mutation. In this experiment, we randomly selected for
sequencing 10 plasmids obtained by transformation of Escherichia coli with the self-ligation mixture, followed by minipreps. All 10 plasmids had a correct mutation with no other alteration in the sequenced open reading frame. Thus, the strength of the protocol described here is that the mutagenesis can be carried out without any reference to restriction sites, with the exception of those introduced into the primers. When deciding which protocol to use, ours or the Phoenix system, the investigator must balance the strength of the procedure described here versus the additional sequencing reactions that may be required. Several criteria must be considered before selecting a Class IIS enzyme for mutagenesis. First, for optimal ligation reactions, there should be several base overhangs in the restriction sites. Second, the cutting site should not be too far from the recognition site. Long separations would make a primer unnecessarily lengthy. We have listed in Table 1 some of the Class IIS restriction enzymes that satisfy these two criteria (see also Ref. 2). The addition of the Class IIS restriction site into the primer limits the number of different Class IIS enzymes the investigator needs in his or her freezer. The same Class IIS restriction site can be generated in any gene. Given the documented efficiency of reconstitution of genes digested with particular Class IIS enzymes, the investigator can use only these effective enzymes and avoid enzymes which may be less efficient in allowing a gene to reassemble (1). Thus, this procedure can be readily applied to any DNA sequence. This strategy does not require primers with mutated nucleotides bordered by annealing sequences both upstream and downstream, which will present a technical challenge if the mutation sequence is very long, as in our case. Therefore, the number of nucleotide changes is only limited by the synthesis of primers. Thus, this strategy is an alternative to commercial kits and the previously reported Phoenix protocol. Acknowledgments. This work was funded in part by NIH Grants CHRC 5 P 30 and 1R01 GM 57427 and by the USDA/ARS under Cooperative Agreement 58-6250-6001. We thank Jon K. Pittman, Ning-hui Cheng, and Coimbatore S. Sreevidya for critical reading of the manuscript.
REFERENCES 1. Berger, S. L., Manrow, R. E., and Lee, H. Y. (1993) Phoenix mutagenesis: One-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Anal. Biochem. 214, 571–579. 2. Berger, S. L. (1994) Expanding the potential of restriction endonucleases: Use of hapaxoterministic enzymes. Anal. Biochem. 222, 1– 8. 3. Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J. C. (1994) Site-directed mutagenesis of doublestranded DNA by the polymerase chain reaction. Gene 151, 119 – 123.
NOTES & TIPS Use of Class IIS Restriction Enzymes for Site-Directed Mutagenesis: Variations on Phoenix Mutagenesis Toshiro Shigaki and Kendal D. Hirschi 1 USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, Texas 77030 Received April 4, 2001; published online September 28, 2001
This report describes a method for creating a mutation in a plasmid without reference to restriction sites. This method requires the synthesis of two primers and PCR to copy an entire supercoiled plasmid. One of the primers contains the mutated region together with a Class IIS restriction site near its 5⬘ end, whereas the other, which is targeted to the same region of the plasmid, bears a recognition site for the same enzyme, also near the 5⬘ end. Upon cleaving the hybridized PCR products with the chosen restriction enzyme, the recognition sites will be jettisoned, and staggered ends that permit seamless joining of the linear products into circular plasmids are created. Hence the primers contain the only required enzyme recognition sites. However, as the entire plasmid is generated by the polymerase chain reaction, it is necessary to sequence all parts of the product in which mistakes of any type cannot be tolerated. Commercially available kits have not removed all troublesome elements from the process of creating mutations in your favorite gene (YFG). 2 Fortunately, an elegantly straightforward technique, which does not require a kit, was developed to circumvent many of the problems (1, 2). In this report we have added variations to that procedure, which is called “phoenix mutagenesis,” to extend its utility. Phoenix mutagenesis makes use of restriction enzymes called hapaxoterministomers that cleave DNA into fragments bearing staggered ends with virtually unique sequences. Hapaxoterministomers are drawn 1 To whom correspondence should be addressed. Fax: (713) 7987078. E-mail: [email protected]. 2 Abbreviations used: YFG, your favorite gene; OFG, our favorite gene.
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from Class IIS restriction enzymes, which cleave outside the recognition sequence, and from enzymes that cleave within interrupted palindromic sequences. Because the overhangs of every fragment so generated are almost always different, each fragment can be joined only to those fragments with which it was originally contiguous. Thus, a plasmid cut with hapaxoterministomers into several fragments can be reborn (hence the name Phoenix) simply by ligating. In such a system, mutations can be created by swapping a fragment of the original template with an engineered fragment bearing both the desired mutation and the ends identical to those of the fragment it was designed to replace. To substitute one fragment for another, the mutated fragment can be added to an unfractionated mixture of the original fragments where it competes with its unmutated couterpart for a place in the finished, circularized plasmid. If the competition is unsuccessful, the superfluous fragment must be removed from the ligation mixture before the mutant counterpart is added. A major advantage of the method is that only the engineered fragment must be sequenced; all others are unchanged. A drawback is that recognition sites for hapaxoterministic enzymes must flank the site of the desired mutation. Our method dispenses altogether with the need for any restriction sites in the target plasmid. Instead, we introduce specific Class IIS restriction sites into the primers used for PCR amplification. This makes the procedure applicable to any gene, regardless of the sequence. The presence of such sites in the plasmid is irrelevant because the final reconstitution of our plasmid occurs in a manner identical with that of the Phoenix system. We believe the development of this modification of the Phoenix protocol is particularly advantageous for the introduction of multiple amino acid changes into YFG. Figure 1 shows in schematic form an example of our mutagenesis approach. In this example, we were interested in altering nine amino acids (27 nucleotides) of our favorite gene (OFG; Fig. 1a). If we were to use commercially available kits, we might first need to subclone the fragment into the appropriate vector; then, after the mutagenesis we would need to shuttle OFG back into the cloning vector for further manipulations. However, utilizing the Phoenix-based variation, we avoid these complications. Initially, a set of Analytical Biochemistry 298, 118 –120 (2001) doi:10.1006/abio.2001.5341 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
119
FIG. 1. An example of site-directed mutagenesis with the Class IIS restriction enzyme BsmBI. (a) The 27 bases of wild-type DNA (upper sequence, underlined) are mutated to the bases indicated by bold letters in the lower sequence. Corresponding amino acid sequences are also shown. (b) Primers used in the mutagenesis. Primer A (the forward primer) consists of the following elements starting at the 5⬘ end. (1) Six random bases for the efficient digestion by BsmBI. (2) BsmBI recognition sequence (boxed). (3) An extra (random) nucleotide as a spacer. This and all other upstream nucleotides are cleaved off by BsmBI. (4) CATT (underlined) will be the overhang created by BsmBI and it will be complementary to the overhang on Primer B. This is not required for annealing as mentioned in the text. (5) Desired mutation sequence as shown in bold letters that is 27 nucleotides long. (6) Twenty-one nucleotides (underlined) that are hybridized to the plasmid template. Primer B (the reverse primer) consists of the following elements starting at the 5⬘ end. (1) Six random bases for efficient digestion. (2) BsmBI recognition sequence (boxed). (3) A random spacer nucleotide. (4) The next four nucleotides, AATG (underlined), both serve as hybridizing sequence and will be a sticky-end overhang when digested with BsmBI. (5) Twenty-one nucleotides (underlined) hybridized to the template. (c) Primers A and B are used to amplify the supercoiled plasmid template to produce an entire, but linearized plasmid. BsmBI sites are shown by a bold line, the introduced mutation sequence is shown by a bold broken line, and the annealing sequences are shown by regular lines parallel to the plasmid template. (d) The double-stranded DNA made by the PCR amplification is shown in detail. Corresponding amino acid sequences are also shown. The linear DNA is digested with BsmBI (the recognition sequence is indicated by a box) to make complementary overhangs at both ends. Gaps have been introduced to show the position of the cleavage sites (small arrows). The digested DNA is then column-purified to remove the small fragments (indicated by italic letters) with a BsmBI recognition sequence. The purified sticky-ended DNA is then simply self-ligated to produce the gene with 27 nucleotide (9 amino acid) long mutations.
primers targeted to the intended mutation site (Fig. 1b) was made in order to amplify the whole plasmid linearly (Fig. 1c). Primer A contains an introduced stretch of nucleotides that are changed to code for the
altered nine amino acids (bold letters, Fig. 1b) upstream of the annealing sequence (underlined, Fig. 1b). At the 5⬘-end of the primer, a BsmBI (a Class IIS enzyme) site was added, along with extra bases (TA-
120
NOTES & TIPS TABLE 1
Type IIS Restriction Enzymes That Can Be Used for Site-Directed Mutagenesis Enzyme BbvII BsmAI BspMI Eco31I BsmBI (Esp3I) FokI HgaI SfaNI Sth132I
Recognition sequence and cleavage site 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘ 5⬘
GAAGACNNˆNNNN_ 3⬘ GTCTCNˆNNNN_ 3⬘ ACCTGCNNNNˆNNNN_ 3⬘ GGTCTCNˆNNNN_ 3⬘ CGTCTCNˆNNNN_ 3⬘ GGATGNNNNNNNNNˆNNNN_ 3⬘ GACGCNNNNNˆNNNNN_ 3⬘ GCATCNNNNNˆNNNN_ 3⬘ CCCGNNNNˆNNNN_ 3⬘
Note. The sequence is for only one strand, and the cleavage site on this strand is indicated by ˆ . The cleavage site on the complementary strand is indicated by _.
GATG) to ensure efficient digestion. The cutting site of BsmBI is one base away from the recognition sequence CGTCTC and five bases away from the complementary recognition sequence GCAGAG. This leaves a fourbase cohesive end within the coding region of OFG. In this example, the sequence of these four bases must originate from the native gene. These bases are complementary to the template, but hybridization to the template is not required for successful mutagenesis. This four-base-pair cohesive end is compatible with the other end amplified at Primer B when digested with the same enzyme (Fig. 1d). We performed PCR in a 100-l volume containing 100 ng of supercoiled plasmid template (4.6 kb), 50 pmol each of forward and reverse primers, 20 nmol dNTP, 1.5 mM MgCl 2, and 2.5 U Expand High Fidelity DNA polymerase (Roche, Indianapolis, IN). The thermocycler was programmed at 94°C for 30 s for denaturation, 58°C for 1 min for annealing, and 68°C for 4 min for elongation, and the cycle was repeated 30 times. The PCR products were column-purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and digested with DpnI to destroy all the wild-type DNAs (the templates for PCR amplification). This restriction enzyme recognizes only dam-methylated DNA such as the plasmid template, leaving the PCRamplified DNA intact (3). Following column purification, the DpnI-digested PCR products were digested with BsmBI and columnpurified again to remove the small DNA fragments where the BsmBI site is located in order to prevent these fragments from competing with the legitimate sticky ends for ligation. The digested fragment was then simply self-ligated at 16°C for 16 h to recreate a seamless coding sequence, but with a large introduced mutation. In this experiment, we randomly selected for
sequencing 10 plasmids obtained by transformation of Escherichia coli with the self-ligation mixture, followed by minipreps. All 10 plasmids had a correct mutation with no other alteration in the sequenced open reading frame. Thus, the strength of the protocol described here is that the mutagenesis can be carried out without any reference to restriction sites, with the exception of those introduced into the primers. When deciding which protocol to use, ours or the Phoenix system, the investigator must balance the strength of the procedure described here versus the additional sequencing reactions that may be required. Several criteria must be considered before selecting a Class IIS enzyme for mutagenesis. First, for optimal ligation reactions, there should be several base overhangs in the restriction sites. Second, the cutting site should not be too far from the recognition site. Long separations would make a primer unnecessarily lengthy. We have listed in Table 1 some of the Class IIS restriction enzymes that satisfy these two criteria (see also Ref. 2). The addition of the Class IIS restriction site into the primer limits the number of different Class IIS enzymes the investigator needs in his or her freezer. The same Class IIS restriction site can be generated in any gene. Given the documented efficiency of reconstitution of genes digested with particular Class IIS enzymes, the investigator can use only these effective enzymes and avoid enzymes which may be less efficient in allowing a gene to reassemble (1). Thus, this procedure can be readily applied to any DNA sequence. This strategy does not require primers with mutated nucleotides bordered by annealing sequences both upstream and downstream, which will present a technical challenge if the mutation sequence is very long, as in our case. Therefore, the number of nucleotide changes is only limited by the synthesis of primers. Thus, this strategy is an alternative to commercial kits and the previously reported Phoenix protocol. Acknowledgments. This work was funded in part by NIH Grants CHRC 5 P 30 and 1R01 GM 57427 and by the USDA/ARS under Cooperative Agreement 58-6250-6001. We thank Jon K. Pittman, Ning-hui Cheng, and Coimbatore S. Sreevidya for critical reading of the manuscript.
REFERENCES 1. Berger, S. L., Manrow, R. E., and Lee, H. Y. (1993) Phoenix mutagenesis: One-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Anal. Biochem. 214, 571–579. 2. Berger, S. L. (1994) Expanding the potential of restriction endonucleases: Use of hapaxoterministic enzymes. Anal. Biochem. 222, 1– 8. 3. Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J. C. (1994) Site-directed mutagenesis of doublestranded DNA by the polymerase chain reaction. Gene 151, 119 – 123.