- Email: [email protected]
Rapid and efficient polymerase chain reaction-based strategies for one-site and two-site mutagenesis
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 331 (2004) 404–406 www.elsevier.com/locate/yabio
Notes & Tips
Rapid and eYcient polymerase chain reaction-based strategies for one-site and two-site mutagenesis夽 Zhilong Gong,a Hai Zhang,a Stephan Gabos,b and Xing-Fang Lia,¤ a
Environmental Health Sciences, Department of Public Health Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2G3 b Health Surveillance Branch, Population Health Division, Alberta Health and Wellness, Telus Plaza North Tower, 10025 Jasper Avenue, Edmonton, Alta., Canada T5J 2N3 Received 5 April 2004 Available online 8 June 2004
PCR-based mutagenesis is routinely used for sitedirected mutagenesis because of its Xexibility and high yield of mutants. A variety of PCR-based mutagenesis protocols have been established to achieve eYcient mutagenesis. Among them, overlap extension [1–4] and megaprimer methods [5–10] appear to be particularly simple and widely used. In a typical overlap extension protocol, two fragments of a target sequence are ampliWed in two separate PCRs by using one universal and one mutagenic primer for each reaction. The two intermediate products have terminal complementarity and can form a new template DNA by duplexing in a second reaction (overlap extension), in which the fused product is ampliWed. To achieve a high accuracy, it is necessary to purify the intermediate products from the Wrst set of PCRs. This is usually carried out by gel electrophoresis [1–4]. The megaprimer method involves two rounds of PCRs requiring one mutagenic primer and two Xanking primers. The Wrst-round PCR is performed using one Xanking primer and the mutagenic primer to generate the “megaprimer,” which is then puriWed and used along with the second Xanking primer for the second-round PCR to produce the Wnal mutant products. While these methods are useful for site-directed mutagenesis, they have some of the following limitations: gel puriWcation required for the Wrst PCR products [5], unwanted ampliWcation of the wildtype template resulting in low mutagenesis eYciency [13], insuYcient or nonspeciWc priming due to the double-stranded megaprimers 夽 Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2004.05.006. ¤ Corresponding author. Fax: +780-492-7800. E-mail address: [email protected] (X.-F. Li).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.05.006
or to the unbalanced melting temperatures of the primers [9], and additional steps required for preparation of the template plasmid [11] or treatment of the PCR products [12]. Improvements have been made to both techniques [11–15]. For example, an improved overlap extension PCR method [11] could achieve 95% eYciency in the generation of mutant DNA. However, this method requires the target DNA to be present in both orientations relative to a universal primer, and the ampliWcation of the wildtype template could not be eliminated. Another improvement to the megaprimer protocol eliminated the gel puriWcation step [12]. However, it introduced several additional manipulations and enzyme treatments of PCR products. Here we describe two strategies (Fig. 1) for one-site and two-site base substitution mutagenesis that present substantial improvements over the current methods. They eliminate the gel puriWcation step, reduce nonspeciWc ampliWcation, and improve accuracy (100% mutant yield). In both strategies, two Xanking primers and two mutagenic primers are designed to have similar melting temperatures. The sequences of the two mutagenic primers used in one-site mutagenesis are complementary but have opposite orientations. For two-site mutagenesis, each of the two mutagenic primers targets one mutagenic site and is paired with an adjacent Xanking primer of opposite orientation. The one-site mutagenesis method is a modiWcation of the overlap extension PCR mutagenesis technique [2]. This method involves two parallel asymmetric PCRs to generate two single stranded mutagenic fragments that are subsequently mixed to perform the second PCR. The Wrst two asymmetric PCRs are carried out in separate tubes with the mutagenic primers having concentrations 1% of the Xanking primers. This step generates two
Notes & Tips / Analytical Biochemistry 331 (2004) 404–406
Fig. 1. Schematic representation of the improved methodology for site-directed mutagenesis. The primers are indicated by arrows, with the mutagenic primers having a vertical bar showing the mutation site. FF, Xanking forward primer; FR, Xanking reverse primer; MF, mutagenic forward primer; MR, mutagenic reverse primer. In the case of one-site mutagenesis, FF and MR generate FFMR (the forward single-stranded mutagenic megaprimer), while MF and FR generate MFFR (the reverse single-stranded mutagenic megaprimer) in the Wrst-round PCRs, carried out in two separate tubes. The two singlestranded mutagenic megaprimers are completely complementary in the mutagenic primers region. In the second-round PCR, the two single-stranded mutagenic megaprimers annealed to each other (A) and are further extended to the full length (B). No denaturation was involved in the second PCR. For two-site mutagenesis, two singlestranded mutagenic primers, FFMR and MFFR, were synthesized in the Wrst-round PCR, same as described above. In the second-round PCR, these two mutagenic megaprimers amplify the full length mutant (C) with the wildtype DNA template of the Wrst-round PCR as the template. A higher annealing temperature (e.g., 72 °C) was used to avoid the annealing of the shorter primers.
overlapped single-stranded mutagenic megaprimers (FFMR and MFFR in Fig. 1). The contents in the two PCR tubes are then mixed to carry out the second PCR, which is a simpliWed process that involves only annealing and extension steps. Because the two single-stranded megaprimers overlap in the entire mutagenic primer region, they can anneal to each other at the annealing step and be extended at the extension step. No denaturation is necessary. The elimination of the denaturation step in the second PCR avoids the dissociation of the wildtype template and the annealing of the Xanking primers to the wildtype template. Therefore, the nonspeciWc ampliWcation of the wildtype template is eliminated, rendering the gel puriWcation step unnecessary. The two-site mutagenesis method is an improved megaprimer PCR mutagenesis technique. Similar to the Wrst PCR in the one-site mutagenesis method, two single-stranded mutagenic megaprimers are Wrst produced in two separate asymmetric PCRs with the mutagenic primers having concentrations 1% of the Xanking primers. After the mixing of these two PCR products, the two single-stranded megaprimers use the same template as that in the Wrst round PCRs to generate the full-length mutant. Because the two megaprimers have much higher melting temperatures than the shorter mutagenic and
405
Xanking primers, a much higher annealing temperature is used in the second PCR to eliminate the ampliWcation of the wildtype template [13]. Therefore, there is no need to purify the products [2] or to enzymatically remove the interfering primers from the Wrst PCRs [12]. The selective and preferential annealing of the mutagenic megaprimers leads to the improved speciWcity and accuracy (mutant yield). In addition, because the two megaprimers produced separately in the Wrst set of PCRs are both single-stranded and their melting temperatures are better matched than those of the shorter mutagenic or Xanking primers and the double-stranded megaprimer that is commonly used in the conventional megaprimer PCR protocol, a higher priming eYciency is achieved. Furthermore, this method is simpler and faster for introducing two-site mutagenesis than the conventional megaprimer PCR mutagenesis methods that require, at a minimum, the generation of a double-stranded mutagenic megaprimer, introduction of a second mutation to the above, and further extension to form a full-length mutant. Having established the methods for one-site and twosite mutagenesis, we have demonstrated an application of the methods to introduce base substitutions into the Cryptosporidium parvum bovine genotype 70-kDa heat shock gene (hsp70). To introduce one base substitution, we have designed two Xanking primers that have melting temperatures of 58.9 and 59.7 °C and two mutagenic primers that have the same melting temperature of 59.1 °C (supplementary Table 1). For each asymmetric PCR, a Xanking primer and a paired mutagenic primer were used to generate a single-stranded megaprimer. The two parallel PCR processes were carried out in separate tubes (50 L), each containing 25 ng template DNA (pBK-CMV plasmid carrying the C. parvum bovine genotype 70-kDa heat shock gene), 15 pmol of the Xanking primer, 0.15 pmol of the mutagenic primer, 15 nmol of each dNTP, 50 nmol of Mg2+, and 2.5 U Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) using the manufacturer-supplied buVer. The reaction solutions were initially heated to 94 °C for 5 min. PCR was performed for 35 cycles to accumulate as much template as possible, under the conditions of 30 s at 94 °C, 30 s at 56.5 °C, and 45 s at 72 °C. After completion of this Wrst round of PCR, the contents of the two reaction mixtures were directly combined to perform the second PCR that involved 25 cycles consisting of 1 min at 56.5 °C and 30 s at 72 °C, with a Wnal extension of 10 min at 72 °C. No additional enzyme and dNTPs were added. No denaturation step was used for the second PCR. The products were directly separated and analyzed using agarose gel electrophoresis (Fig. 2). The predominant band at 732 bp indicates the presence (195%) of the expected mutagenic product. We have further introduced two base substitutions to hsp70 of C. parvum (bovine genotype) using the two-site
406
Notes & Tips / Analytical Biochemistry 331 (2004) 404–406
could be made to extend the methods to include base insertion and deletion mutagenesis.
Acknowledgments
Fig. 2. Agarose gel electrophoresis showing the one-site and two-site mutagenesis products. An aliquot of 5 L of the PCR products (without any puriWcation) was mixed with 5 L of 2X loading buVer and was subsequently loaded onto the gel. The bottom lane is the 100-bp DNA molecular weight ladder, the middle lane is the product of onesite mutagenesis, and the top lane is the two-site mutagenesis product.
This work was supported by Grants from the Natural Sciences and Engineering Research Council of Canada and Alberta Health and Wellness. NSERC is also acknowledged for the University Faculty Award to X.F.L.
References mutagenesis method. The Xanking primers were the same as those for one-site mutagenesis, and the two mutagenic primers that we designed have melting temperatures of 59.1 and 59.4 °C (supplementary Table 1). One PCR involved the forward Xanking primer and the paired reverse mutagenic primer while the other PCR used the reverse Xanking primer and the paired forward mutagenic primer. The conditions for the Wrst-round PCR were the same as those for the one-site mutagenesis. The contents of the PCR were combined, and a twostep PCR consisting of 25 cycles of 30 s at 94 °C and 1 min at 72 °C with a Wnal extension at 72 °C for 10 min followed. Because the two single-stranded megaprimers generated from the Wrst-round PCR were 189 and 512 nt in length with an estimated melting temperature higher than 77 °C, the annealing and the extension steps were combined and both performed at 72 °C. Direct analysis of the reaction mixture using agarose gel electrophoresis (Fig. 2) shows the presence of the desired products (732 bp) as the predominant (180%) product. The obtained mutants for both one-site and two-site mutagenesis were further cloned using Zero Blunt PCR Cloning Kit (Invitrogen, Carlsbad, CA). Ten colonies for each mutagenesis experiment were selected. The collected plasmids were restriction digested, and they all showed the existence of the inserts. These plasmids were then sequenced to conWrm the existence of the mutations introduced. The expected base substitutions were conWrmed in all 20 colonies, and no extra mutations were present, demonstrating the excellent accuracy (100% mutant yield) of the methods. The improved mutagenesis methods reduce the nonspeciWc priming, avoid the ampliWcation of the wildtype template, eliminate the gel puriWcation step, and enhance the accuracy and product yield. They are simple and easy to use. There is no special requirement for the wildtype template. The methods can in principle be applied to any one-site and two-site mutagenesis. Although the methods were designed and tested only for base substitutions, further modiWcations with appropriate primer designs
[1] R. Higuchi, B. Krummel, R.K. Saiki, A general method of in vitro preparation and speciWc mutagenesis of DNA fragments: study of protein and DNA interactions, Nucleic Acids Res. 16 (1988) 7351– 7367. [2] S.N. Ho, R.M. Horton, J.K. Pullen, L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77 (1989) 51–59. [3] M.D. Davis, R.S. Wonderling, S.L. Walker, R.A. Owens, Analysis of the eVects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro, J. Virol. 73 (1999) 2084–2093. [4] R.M. Jones, P.A. Williams, Mutational analysis of the critical bases involved in activation of the AreR-regulated sigma54dependent promoters in Acinetobacter sp. strain ADP1, Appl. Environ. Microbiol. 69 (2003) 5627–5635. [5] M. Kammann, J. Laufs, J. Schell, B. Gronenborn, Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res. 17 (1989) 5404. [6] G. Sarkar, S.S. Sommer, The megaprimer method of site-directed mutagenesis, Biotechniques 8 (1990) 404–407. [7] G. Sarkar, S.S. Sommer, Double-stranded DNA segments can eYciently prime the ampliWcation of human genomic DNA, Nucleic Acids Res. 20 (1992) 4937–4938. [8] O. Landt, H.-P. Grunert, U. Hahn, A general method for rapid site-directed mutagenesis using the polymerase chain reaction, Gene 96 (1990) 125–128. [9] A.M. Smith, K.P. Klugman, “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor, Biotechniques 22 (1997) 438–442. [10] S. Angelaccio, M.C. Bonaccorsi di Patti, Site-directed mutagenesis by the megaprimer PCR method: variations on a theme for simultaneous introduction of multiple mutations, Anal. Biochem. 306 (2002) 346–349. [11] A. Urban, S. Nenkirchen, K.-E. Jaeger, A rapid and eYcient method for site-directed mutagenesis using one- step overlap extension PCR, Nucleic Acids Res. 25 (1997) 2227–2228. [12] B. Seraphin, S. Kandels-Lewis, An eYcient PCR mutagenesis strategy without gel puriWcation [correction of puriWciation] step that is amenable to automation, Nucleic Acids Res. 24 (1996) 3276–3277. [13] S.-H. Ke, E.L. Madison, Rapid and eYcient site-directed mutagenesis by single-tube “megaprimer” PCR method, Nucleic Acids Res. 25 (1997) 3371–3372. [14] J. Brons-Poulsen, N.E. Petersen, M. Horder, K. Kristiansen, An improved PCR-based method for site directed mutagenesis using megaprimers, Mol. Cell. Probes 12 (1998) 345–348. [15] L. Ge, P. Rudolph, Simultaneous introduction of multiple mutations using overlap extension PCR, Biotechniques 22 (1997) 28–30.
Notes & Tips
Rapid and eYcient polymerase chain reaction-based strategies for one-site and two-site mutagenesis夽 Zhilong Gong,a Hai Zhang,a Stephan Gabos,b and Xing-Fang Lia,¤ a
Environmental Health Sciences, Department of Public Health Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2G3 b Health Surveillance Branch, Population Health Division, Alberta Health and Wellness, Telus Plaza North Tower, 10025 Jasper Avenue, Edmonton, Alta., Canada T5J 2N3 Received 5 April 2004 Available online 8 June 2004
PCR-based mutagenesis is routinely used for sitedirected mutagenesis because of its Xexibility and high yield of mutants. A variety of PCR-based mutagenesis protocols have been established to achieve eYcient mutagenesis. Among them, overlap extension [1–4] and megaprimer methods [5–10] appear to be particularly simple and widely used. In a typical overlap extension protocol, two fragments of a target sequence are ampliWed in two separate PCRs by using one universal and one mutagenic primer for each reaction. The two intermediate products have terminal complementarity and can form a new template DNA by duplexing in a second reaction (overlap extension), in which the fused product is ampliWed. To achieve a high accuracy, it is necessary to purify the intermediate products from the Wrst set of PCRs. This is usually carried out by gel electrophoresis [1–4]. The megaprimer method involves two rounds of PCRs requiring one mutagenic primer and two Xanking primers. The Wrst-round PCR is performed using one Xanking primer and the mutagenic primer to generate the “megaprimer,” which is then puriWed and used along with the second Xanking primer for the second-round PCR to produce the Wnal mutant products. While these methods are useful for site-directed mutagenesis, they have some of the following limitations: gel puriWcation required for the Wrst PCR products [5], unwanted ampliWcation of the wildtype template resulting in low mutagenesis eYciency [13], insuYcient or nonspeciWc priming due to the double-stranded megaprimers 夽 Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2004.05.006. ¤ Corresponding author. Fax: +780-492-7800. E-mail address: [email protected] (X.-F. Li).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.05.006
or to the unbalanced melting temperatures of the primers [9], and additional steps required for preparation of the template plasmid [11] or treatment of the PCR products [12]. Improvements have been made to both techniques [11–15]. For example, an improved overlap extension PCR method [11] could achieve 95% eYciency in the generation of mutant DNA. However, this method requires the target DNA to be present in both orientations relative to a universal primer, and the ampliWcation of the wildtype template could not be eliminated. Another improvement to the megaprimer protocol eliminated the gel puriWcation step [12]. However, it introduced several additional manipulations and enzyme treatments of PCR products. Here we describe two strategies (Fig. 1) for one-site and two-site base substitution mutagenesis that present substantial improvements over the current methods. They eliminate the gel puriWcation step, reduce nonspeciWc ampliWcation, and improve accuracy (100% mutant yield). In both strategies, two Xanking primers and two mutagenic primers are designed to have similar melting temperatures. The sequences of the two mutagenic primers used in one-site mutagenesis are complementary but have opposite orientations. For two-site mutagenesis, each of the two mutagenic primers targets one mutagenic site and is paired with an adjacent Xanking primer of opposite orientation. The one-site mutagenesis method is a modiWcation of the overlap extension PCR mutagenesis technique [2]. This method involves two parallel asymmetric PCRs to generate two single stranded mutagenic fragments that are subsequently mixed to perform the second PCR. The Wrst two asymmetric PCRs are carried out in separate tubes with the mutagenic primers having concentrations 1% of the Xanking primers. This step generates two
Notes & Tips / Analytical Biochemistry 331 (2004) 404–406
Fig. 1. Schematic representation of the improved methodology for site-directed mutagenesis. The primers are indicated by arrows, with the mutagenic primers having a vertical bar showing the mutation site. FF, Xanking forward primer; FR, Xanking reverse primer; MF, mutagenic forward primer; MR, mutagenic reverse primer. In the case of one-site mutagenesis, FF and MR generate FFMR (the forward single-stranded mutagenic megaprimer), while MF and FR generate MFFR (the reverse single-stranded mutagenic megaprimer) in the Wrst-round PCRs, carried out in two separate tubes. The two singlestranded mutagenic megaprimers are completely complementary in the mutagenic primers region. In the second-round PCR, the two single-stranded mutagenic megaprimers annealed to each other (A) and are further extended to the full length (B). No denaturation was involved in the second PCR. For two-site mutagenesis, two singlestranded mutagenic primers, FFMR and MFFR, were synthesized in the Wrst-round PCR, same as described above. In the second-round PCR, these two mutagenic megaprimers amplify the full length mutant (C) with the wildtype DNA template of the Wrst-round PCR as the template. A higher annealing temperature (e.g., 72 °C) was used to avoid the annealing of the shorter primers.
overlapped single-stranded mutagenic megaprimers (FFMR and MFFR in Fig. 1). The contents in the two PCR tubes are then mixed to carry out the second PCR, which is a simpliWed process that involves only annealing and extension steps. Because the two single-stranded megaprimers overlap in the entire mutagenic primer region, they can anneal to each other at the annealing step and be extended at the extension step. No denaturation is necessary. The elimination of the denaturation step in the second PCR avoids the dissociation of the wildtype template and the annealing of the Xanking primers to the wildtype template. Therefore, the nonspeciWc ampliWcation of the wildtype template is eliminated, rendering the gel puriWcation step unnecessary. The two-site mutagenesis method is an improved megaprimer PCR mutagenesis technique. Similar to the Wrst PCR in the one-site mutagenesis method, two single-stranded mutagenic megaprimers are Wrst produced in two separate asymmetric PCRs with the mutagenic primers having concentrations 1% of the Xanking primers. After the mixing of these two PCR products, the two single-stranded megaprimers use the same template as that in the Wrst round PCRs to generate the full-length mutant. Because the two megaprimers have much higher melting temperatures than the shorter mutagenic and
405
Xanking primers, a much higher annealing temperature is used in the second PCR to eliminate the ampliWcation of the wildtype template [13]. Therefore, there is no need to purify the products [2] or to enzymatically remove the interfering primers from the Wrst PCRs [12]. The selective and preferential annealing of the mutagenic megaprimers leads to the improved speciWcity and accuracy (mutant yield). In addition, because the two megaprimers produced separately in the Wrst set of PCRs are both single-stranded and their melting temperatures are better matched than those of the shorter mutagenic or Xanking primers and the double-stranded megaprimer that is commonly used in the conventional megaprimer PCR protocol, a higher priming eYciency is achieved. Furthermore, this method is simpler and faster for introducing two-site mutagenesis than the conventional megaprimer PCR mutagenesis methods that require, at a minimum, the generation of a double-stranded mutagenic megaprimer, introduction of a second mutation to the above, and further extension to form a full-length mutant. Having established the methods for one-site and twosite mutagenesis, we have demonstrated an application of the methods to introduce base substitutions into the Cryptosporidium parvum bovine genotype 70-kDa heat shock gene (hsp70). To introduce one base substitution, we have designed two Xanking primers that have melting temperatures of 58.9 and 59.7 °C and two mutagenic primers that have the same melting temperature of 59.1 °C (supplementary Table 1). For each asymmetric PCR, a Xanking primer and a paired mutagenic primer were used to generate a single-stranded megaprimer. The two parallel PCR processes were carried out in separate tubes (50 L), each containing 25 ng template DNA (pBK-CMV plasmid carrying the C. parvum bovine genotype 70-kDa heat shock gene), 15 pmol of the Xanking primer, 0.15 pmol of the mutagenic primer, 15 nmol of each dNTP, 50 nmol of Mg2+, and 2.5 U Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) using the manufacturer-supplied buVer. The reaction solutions were initially heated to 94 °C for 5 min. PCR was performed for 35 cycles to accumulate as much template as possible, under the conditions of 30 s at 94 °C, 30 s at 56.5 °C, and 45 s at 72 °C. After completion of this Wrst round of PCR, the contents of the two reaction mixtures were directly combined to perform the second PCR that involved 25 cycles consisting of 1 min at 56.5 °C and 30 s at 72 °C, with a Wnal extension of 10 min at 72 °C. No additional enzyme and dNTPs were added. No denaturation step was used for the second PCR. The products were directly separated and analyzed using agarose gel electrophoresis (Fig. 2). The predominant band at 732 bp indicates the presence (195%) of the expected mutagenic product. We have further introduced two base substitutions to hsp70 of C. parvum (bovine genotype) using the two-site
406
Notes & Tips / Analytical Biochemistry 331 (2004) 404–406
could be made to extend the methods to include base insertion and deletion mutagenesis.
Acknowledgments
Fig. 2. Agarose gel electrophoresis showing the one-site and two-site mutagenesis products. An aliquot of 5 L of the PCR products (without any puriWcation) was mixed with 5 L of 2X loading buVer and was subsequently loaded onto the gel. The bottom lane is the 100-bp DNA molecular weight ladder, the middle lane is the product of onesite mutagenesis, and the top lane is the two-site mutagenesis product.
This work was supported by Grants from the Natural Sciences and Engineering Research Council of Canada and Alberta Health and Wellness. NSERC is also acknowledged for the University Faculty Award to X.F.L.
References mutagenesis method. The Xanking primers were the same as those for one-site mutagenesis, and the two mutagenic primers that we designed have melting temperatures of 59.1 and 59.4 °C (supplementary Table 1). One PCR involved the forward Xanking primer and the paired reverse mutagenic primer while the other PCR used the reverse Xanking primer and the paired forward mutagenic primer. The conditions for the Wrst-round PCR were the same as those for the one-site mutagenesis. The contents of the PCR were combined, and a twostep PCR consisting of 25 cycles of 30 s at 94 °C and 1 min at 72 °C with a Wnal extension at 72 °C for 10 min followed. Because the two single-stranded megaprimers generated from the Wrst-round PCR were 189 and 512 nt in length with an estimated melting temperature higher than 77 °C, the annealing and the extension steps were combined and both performed at 72 °C. Direct analysis of the reaction mixture using agarose gel electrophoresis (Fig. 2) shows the presence of the desired products (732 bp) as the predominant (180%) product. The obtained mutants for both one-site and two-site mutagenesis were further cloned using Zero Blunt PCR Cloning Kit (Invitrogen, Carlsbad, CA). Ten colonies for each mutagenesis experiment were selected. The collected plasmids were restriction digested, and they all showed the existence of the inserts. These plasmids were then sequenced to conWrm the existence of the mutations introduced. The expected base substitutions were conWrmed in all 20 colonies, and no extra mutations were present, demonstrating the excellent accuracy (100% mutant yield) of the methods. The improved mutagenesis methods reduce the nonspeciWc priming, avoid the ampliWcation of the wildtype template, eliminate the gel puriWcation step, and enhance the accuracy and product yield. They are simple and easy to use. There is no special requirement for the wildtype template. The methods can in principle be applied to any one-site and two-site mutagenesis. Although the methods were designed and tested only for base substitutions, further modiWcations with appropriate primer designs
[1] R. Higuchi, B. Krummel, R.K. Saiki, A general method of in vitro preparation and speciWc mutagenesis of DNA fragments: study of protein and DNA interactions, Nucleic Acids Res. 16 (1988) 7351– 7367. [2] S.N. Ho, R.M. Horton, J.K. Pullen, L.R. Pease, Site-directed mutagenesis by overlap extension using the polymerase chain reaction, Gene 77 (1989) 51–59. [3] M.D. Davis, R.S. Wonderling, S.L. Walker, R.A. Owens, Analysis of the eVects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro, J. Virol. 73 (1999) 2084–2093. [4] R.M. Jones, P.A. Williams, Mutational analysis of the critical bases involved in activation of the AreR-regulated sigma54dependent promoters in Acinetobacter sp. strain ADP1, Appl. Environ. Microbiol. 69 (2003) 5627–5635. [5] M. Kammann, J. Laufs, J. Schell, B. Gronenborn, Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR), Nucleic Acids Res. 17 (1989) 5404. [6] G. Sarkar, S.S. Sommer, The megaprimer method of site-directed mutagenesis, Biotechniques 8 (1990) 404–407. [7] G. Sarkar, S.S. Sommer, Double-stranded DNA segments can eYciently prime the ampliWcation of human genomic DNA, Nucleic Acids Res. 20 (1992) 4937–4938. [8] O. Landt, H.-P. Grunert, U. Hahn, A general method for rapid site-directed mutagenesis using the polymerase chain reaction, Gene 96 (1990) 125–128. [9] A.M. Smith, K.P. Klugman, “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor, Biotechniques 22 (1997) 438–442. [10] S. Angelaccio, M.C. Bonaccorsi di Patti, Site-directed mutagenesis by the megaprimer PCR method: variations on a theme for simultaneous introduction of multiple mutations, Anal. Biochem. 306 (2002) 346–349. [11] A. Urban, S. Nenkirchen, K.-E. Jaeger, A rapid and eYcient method for site-directed mutagenesis using one- step overlap extension PCR, Nucleic Acids Res. 25 (1997) 2227–2228. [12] B. Seraphin, S. Kandels-Lewis, An eYcient PCR mutagenesis strategy without gel puriWcation [correction of puriWciation] step that is amenable to automation, Nucleic Acids Res. 24 (1996) 3276–3277. [13] S.-H. Ke, E.L. Madison, Rapid and eYcient site-directed mutagenesis by single-tube “megaprimer” PCR method, Nucleic Acids Res. 25 (1997) 3371–3372. [14] J. Brons-Poulsen, N.E. Petersen, M. Horder, K. Kristiansen, An improved PCR-based method for site directed mutagenesis using megaprimers, Mol. Cell. Probes 12 (1998) 345–348. [15] L. Ge, P. Rudolph, Simultaneous introduction of multiple mutations using overlap extension PCR, Biotechniques 22 (1997) 28–30.