Promega Notes Magazine Number 62, 1997, p. 02 The GeneEditor TM in vitro Mutagenesis System: Site- Directed Mutagenesis Using Altered Beta-Lactamase Specificity By Christine Andrews and Scott Lesley Promega Corporation Promega's new GeneEditor TM in vitro Site-Directed Mutagenesis System * provides a high efficiency, reliable procedure for the generation and selection of oligonucleotide-directed mutants. The Selection s provided in this system encode mutations which alter the substrate specificity of beta-lactamase, the gene encoding ampicillin resistance, commonly found on cloning vectors. This altered substrate specificity provides a positive selection for the mutant strand and results in consistently high mutagenesis frequencies using either double-stranded (ds) or single-stranded (ss) DNA templates. * Patent Pending. Introduction Site-directed mutagenesis is a valuable tool for the study of DNA function and protein structure and function. A number of different mutagenesis methods have been reported (1,2). Site-directed in vitro mutagenesis (3) is accomplished by hybridizing a target DNA to a synthetic oligonucleotide which is complementary to the target template except for a region of mismatch near the center. It is this region that contains the desired mutation. Following hybridization, the oligonucleotide is extended with DNA polymerase to create a doublestranded structure. The nick is then sealed with DNA ligase and the duplex structure is transformed into an E. coli host. The GeneEditor TM in vitro Site-Directed Mutagenesis System uses antibiotic resistance to select for plasmid derived from the mutant strand. This positive selection results in consistently high mutagenesis efficiencies (often >90%). If no selection method for mutants is employed, the theoretical yield of mutants using this procedure is 50% (due to the semi-conservative mode of DNA replication). In practice, however, the mutant yield in the absence of selection may be much lower, often only a few percent or less. This is assumed to be due to such factors as incomplete in vitro polymerization, primer displacement by the DNA polymerase used in the fill-in reaction, and in vivo host-directed mismatch repair mechanisms which favor repair of the newly synthesized unmethylated DNA strand (4). Positive selection of the mutant strand The GeneEditor TM System (Figure 1) alters ampicillin resistance to provide a selection which is specific to the newly synthesized mutant strand. Beta-lactam antibiotics, such as ampicillin, cause cell death in E. coli by inhibiting cell wall formation. Beta-lactamases confer resistance to ampicillin and other beta-lactam antibiotics (i.e., penicillins and cephalosporins) by degrading these compounds to an inactive form. The beta-lactamase enzyme inactivates these antibiotics by hydrolyzing the beta-lactam bond common to all penicillin and cephalosporin derivatives. These two broad classes of antibiotics contain many variations to the basic structure shown in Figure 2. The ability of beta-lactamase enzymes to recognize and degrade a variety of beta-lactam antibiotics has been well characterized (5). The wild-type TEM-1 beta-lactamase found on most cloning vectors is proficient at degrading antibiotics such as ampicillin. Cells expressing this beta-lactamase are therefore resistant to ampicillin. This resistance is used as a means of selection for the maintenance of most common cloning vectors. The TEM-1 enzyme is much less proficient at degrading some other beta-lactams, and cells containing the wild-type enzyme are sensitive to moderate levels of these antibiotics.
Figure 1. Schematic diagram of the GeneEditor TM in vitro Site-Directed Mutagenesis procedure. Figure 2. Structure of beta-lactam antibiotics. Numerous mutations which alter the substrate specificity of the beta-lactamase enzyme have been identified. Cells harboring these mutations exhibit increased resistance to a broader spectrum of beta-lactam antibiotics while retaining resistance to ampicillin (6). These mutations provide a means to select for the mutant strand and are the basis for the high level of efficiency of the GeneEditor TM System. The GeneEditor TM in vitro Site-Directed Mutagenesis System The GeneEditor TM in vitro Site-Directed Mutagenesis System may be used with any vector containing an ampicillin resistance gene and the gene of interest. Figure 1 outlines the mutagenesis procedure, which can be performed using either dsdna or ssdna templates. A Selection provided with the system is annealed to the DNA template at the same time as the mutagenic oligonucleotide. Both the Selection and the mutagenic oligonucleotide must hybridize to the same strand and are used to prime synthesis of the mutant strand. Subsequent synthesis and ligation of this mutant strand links the two oligonucleotides. The mutagenesis reaction is transformed initially into a repair minus (muts) E. coli strain (BMH 71-18) to avoid repair of the desired mutation. The muts mutation prevents the formation of a repair complex at the point of the mismatch between the wild-type and mutant strand. The GeneEditor TM Site-Directed Mutagenesis System uses a proprietary mixture of antibiotics (the GeneEditor TM Antibiotic Selection Mix) to select only those cells containing the desired mutation. A subsequent strain transfer into E. coli JM109 and growth in media containing the GeneEditor TM Antibiotic Selection Mix ensures proper segregation of mutant and wild-type plasmids and results
in a high proportion of mutants. Mutants can be maintained using ampicillin alone for subsequent procedures. The GeneEditor TM System provides BMH 71-18 and JM109 Competent Cells and the GeneEditor TM Antibiotic Selection Mix, as well as the required enzymes, buffers, and Selection s. A variety of bacterial strains were tested for sensitivity to the antibiotics used for selection. All of the strains used, including BMH 71-18 (muts), ES1301 (muts), JM109, HB101, DH5alpha (Life Technologies, Inc.), XL1-Blue and SURE (Stratagene) showed the appropriate increase in resistance to the antibiotic when transformed with mutant DNA generated using the GeneEditor TM System. Making difficult mutations using the GeneEditor TM System Single base changes and other small mutations are the most common types of mutations created using conventional site-directed mutagenesis techniques. The generation of large insertions or deletions is often restricted by the intrinsic properties of the oligonucleotide primer and the target sequence. Base composition and formation of secondary structure in large primers can influence efficient hybridization to the target, and optimization of annealing conditions is often required. Suggestions for mutagenic oligonucleotide design and locations of additional resources are provided in reference 7. Positive selection of mutants with the GeneEditor TM System provides advantages over other site-directed mutagenesis systems. The antibiotic selection is efficient, simple to use and requires little DNA manipulation or hands-on time. The system is compatible with most popular cloning vectors and no subcloning is required. There also is no requirement for unique restriction sites or reliance on the efficiency of the restriction enzyme. Double-stranded template DNA can be used and there is no need to grow the template in a specialized bacterial strain. The T4 DNA Polymerase supplied with the system has high fidelity of incorporation and shows no strand displacement. This results in efficient incorporation of mutagenic oligonucleotides and less risk of undesirable secondary mutations than methods using other DNA polymerases. Various oligonucleotides were designed to assess the mutagenesis efficiencies of different types of mutations, including large insertions and deletions, using the GeneEditor TM System (Table 1). For large insertions and deletions, oligonucleotides were designed to include 20 perfectly matched base pairs on either side of the mutagenic region. To compare efficiencies using different types of DNA template, the GeneEditor TM System in vitro mutagenesis procedure was performed using both dsdna and ssdna derived from several different vectors. All the plasmid templates that were used contain the TEM-1 beta-lactamase gene. Mutagenic oligonucleotides were hybridized at a 25:1 oligonucleotide:template ratio, cooling from 75-37 C at 1.5 C per minute using a thermal cycler. The percent efficiency of mutagenesis reactions for both dsdna and ssdna templates using each of these oligonucleotides and the GeneEditor TM System are shown in Table 1. Table 1. Mutations Generated Using the Gene Editor TM System. Mutagenic Vector Size Type of Mutation A pgem ** -3Zf(+) 32mer 4 base pair deletion in LacZ alphapeptide Efficiency dsdna template (%) 83% Efficiency ssdna template (%) B pgem -3Zf(+) 90mer 50 base pair insertion 59% 78% C pgem -3Zf (+) 40mer 50 base pair deletion 96% 87% D pgem -3Zf(+) 40mer 700 base pair deletion 64% 48% E pbr322 27mer 4 base pair deletion in Tet r gene 71-100% G pgem -7Zf(+) 5 oligos oligos encoding deletions and substitutions F pgem -11Zf(+) 23mer 4 base pair deletion in LacZ alphapeptide 73-75% 30% contain all 5 mutations H pkk223 41mer substitution encoding 2 amino acids 80% I pgem -4Z 42mer multiple substitutions 80-100% Clones were screened by blue/white screening, antibiotic resistance or by restriction digestion and were confirmed by size. to template molar
ratios were 25:1 as recommended in the GeneEditor TM in vitro Site-Directed Mutagenesis System Technical Manual #TM047. Hybridization conditions were 75 C for 5 minutes, followed by cooling to 37 C at a rate of 1.5 C per minute in a thermal cycler. **U.S. Pat. No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted. Results Mutation rates between 48% and 96% were obtained for all the single mutations created using the GeneEditor TM System. Comparable efficiencies were achieved using dsdna and ssdna templates. Thus, for most mutations, preparation of ssdna is not necessary. The results shown in Table 2 demonstrate linkage of the Selection with the mutagenic oligonucleotide. In this experiment, mutagenic oligonucleotides that induce a frameshift mutation in the LacZ alpha-peptide corresponding to the top or bottom strand were used with either the top or bottom strand Selection s using double-stranded pgem -11Zf(+) Vector DNA as a template. The results clearly show linkage of the selection and mutagenic oligonucleotides when hybridized to the same strand and demonstrate the power of the antibiotic selection in providing efficient mutagenesis. Mutagenic oligonucleotides can be used which hybridize to either DNA strand providing the correct Selection is used. Table 2. Linkage of Selection and Mutagenic s. Mutagenic Selection % Mutants bottom strand bottom strand 75 bottom strand top strand <1 top strand bottom strand <1 top strand top strand 73 Figure 3 shows the results of a typical mutant screen using restriction analysis. Mutants generated using oligonucleotide D (Table 1) were grown overnight in the presence of the GeneEditor TM Antibiotic Selection Mix. DNA from each clone was isolated and digested with Nde I and Pvu II. Digestion of the wild-type vector with these enzymes resulted in the generation of two bands of 580bp and 399bp (lane 3). The deletion of the 700bp target sequence in the mutant resulted in the production of a 279bp band (lanes 4, 6-8 and 10). In this example, 14 of a total of 22 colonies screened were positive for the deletion. In addition to single mutations, such as those shown in Figure 3, multiple mutations have also been created using the GeneEditor TM System. Multiple mutations can be generated by using more than one mutagenic oligonucleotide. We have introduced five separate mutagenic oligonucleotides encoding novel restriction sites into the pgem -7Zf(+) Vector with 30% efficiency using this system (Table 1.G). Figure 3. Nde I/Pvu II restriction digests on dsdna template, 700 base-pair deletion mutants. DNA from individual clones was prepared from 3ml cultures using Promega's Wizard Plus Minipreps DNA Purification System. Five microliters of each miniprep was incubated with 5 units each of Nde I and Pvu II at 37 C for 60 minutes. DNA fragments were separated on a 4-20% Tris-Glycine acrylamide gel and post-stained with ethidium bromide. Lanes: lane 1, pgem DNA Markers (Cat.# G1741); lane 2, uncut pgem -3Zf(+) Vector; lane 3, Nde I and Pvu II digest of wild-type pgem - 3Zf(+) Vector; lanes 4-10, Nde I and Pvu II digests of individual clone DNA. The deletion of a 700bp target sequence in the mutated DNA results in the production of a 279bp Nde I and Pvu II fragment. Nde I and Pvu II digestion of wild-type DNA results in the production of two fragments of 580bp and 399bp, respectively. Summary The GeneEditor TM in vitro Site-Directed Mutagenesis System offers several advantages over conventional approaches to oligonucleotide-directed mutagenesis. Like Promega's Altered-Sites II in vitro Mutagenesis System, the GeneEditor TM System relies on positive antibiotic selection of the mutant strand. This approach consistently results in a high proportion of mutants. Unlike other mutagenesis systems, the GeneEditor TM System can be used with most common cloning vectors that contain an ampicillin resistance gene, without subcloning into a specialized vector. The system uses the high fidelity T4 DNA Polymerase to reduce unwanted
secondary mutations. Difficult mutations can be generated using dsdna templates without extensive optimization of hybridization conditions. High mutagenesis efficiencies are consistently and easily achieved using the GeneEditor TM System. References 1. Smith, M. (1985) Ann. Rev. Genet. 19, 423. 2. Site-Specific Mutagenesis and Protein Engineering, Section IV, Chapters 17-21 (1987) Meth. Enzymol. 154, 329. 3. Hutchinson, C.A. et al. (1978) J. Biol. Chem. 253, 6551. 4. Kramer, B., Kramer, W. and Fritz, H.J. (1984) Cell 38, 897. 5. Matagne, A. et al. (1990) Biochem. J. 265, 131. 6. Venkatachalam, K.V. et al. (1994) J. Biol. Chem. 269, 23444. 7. Cosby, N.C. and Lesley, S. (1997) Promega Notes 61, 12. Ordering Information Product GeneEditor TM in vitro Site-Directed Mutagenesis System Cat.# Q9280 1997 Promega Corporation. All Rights Reserved. Altered Sites, pgem and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. GeneEditor is a trademark of Promega Corporation. DH5alpha is a registered trademark of Life Technologies, Inc. SURE is a registered trademark of Stratagene.