Mutants Made Easy

Suppliers of in vitro Site-directed Mutagenesis Kits Biological research greatly benefits from the ability to introduce specific mutations into a DNA sequence. Researchers use site-directed mutagenesis procedures to precisely analyze individual amino acid residues in a protein sequence and in specific protein-nucleic acid interactions. Likewise, serial deletion and random insertion protocols can ease protein structural studies and promoter analyses. In their original incarnations, site-directed mutagenesis protocols required the isolation of single-stranded DNA (ssDNA) from bacteriophage (bacterial viruses), followed by annealing of a primer that was complementary to the target DNA except at the site to be mutated, and the synthesis of a complete "mutant" second strand by a DNA polymerase. Finally, the resultant heteroduplex plasmids were ligated and transformed into bacteria. With no additional enrichments, this protocol theoretically yields a 50 percent mutagenesis frequency; in practice, however, the rate is much lower and results in the need to screen many potential mutants. These old protocols suffer from a number of other drawbacks, most notably, the need to have the target DNA in specific vectors and the difficulty inherent in isolating high-quality ssDNA. Fortunately, current technology provides a range of options for researchers who want to make specific mutations. Some of these protocols require that the DNA of interest be cloned in specific vectors, some require specialized bacterial strains, and still others provide even greater flexibility. Given all the available choices, most likely there is a system that will meet any given requirement and intended application. This profile will primarily focus on kits that enable site-directed mutagenesis, but progressive deletion and transposon-based insertion methods will also be discussed. Classics Never Die Although most of the mutagenesis kits reviewed here use alternative means, a few companies still supply kits based on the rescue of ssDNA template from modified phage (such as M13 or f1), or from phagemids. Phagemids are plasmids that contain M13 or f1 replication origins, enabling the DNA to be packaged in virion particles as ssDNA upon infection of the cells with a helper virus. The Muta-Gene(tm) Kit from Bio-Rad of Hercules, Calif., and the Mutan(tm)-K Site-Directed Mutagenesis System from PanVera of Madison, Wis., both employ the Kunkel mutagenesis protocol.1,2 Investigators collect ssDNA containing the sequence to be mutated from a bacterial strain carrying mutations in both dUTPase (dut) and uracil-N-glycosylase (ung); these strains occasionally substitute uracil for thymine in newly synthesized DNA. After annealing a mutant primer to the ssDNA and synthesizing a new DNA strand, the plasmid is transformed into an ung+ strain that will digest the parental strand of the transformed heteroduplex because it contains uracil residues, enriching for the mutant strand. A number of companies have adopted alternative strategies to ease the difficulties encountered in isolating high-quality ssDNA. La Jolla, Calif.-based Stratagene's QuikChange(tm) Mutagenesis Kit employs a simple four-step procedure in which two primers that are complementary to opposite strands of the target DNA are extended using PfuTurbo(tm) DNA polymerase, a high-fidelity enzyme mixture that minimizes unwanted mutations. The primers are designed such that the resulting mutated plasmid contains staggered nicks; thus, DNA synthesis always uses the wild-type plasmid as a template, and not the newly synthesized strands. The parental DNA template is eliminated by incubation with DpnI, which digests DNA methylated at the sequence 5'-Gm6ATC-3'. Because DNA isolated from Escherichia coli is almost always methylated, but DNA synthesized in vitro is not, this step enriches for the mutant plasmids. The resulting nicked DNA is repaired when transformed into E. coli. That this approach can be carried out in any plasmid, with no subcloning, is one of Quik Change's primary advantages, according to Fernando Macian, a postdoctoral fellow at the Center for Blood Research in Boston. For extra-long templates, Stratagene also offers the QuikChange XL kit. Three commercial offerings based on the Unique Site Elimination technique of W.P. Deng and J.A. Nikoloff3 are the U.S.E. Site-Directed Mutagenesis Kit from Amersham Pharmacia Biotech of Piscataway, N.J.; the Chameleon(r) Double-Stranded, Site-Directed Mutagenesis Kit from Stratagene; and the Transformer(tm) Site-Directed Mutagenesis Kit from CLONTECH of Palo Alto, Calif. The U.S.E. protocol uses two primers: one to generate a mutation in the sequence of interest, and the other to introduce a mutation in a nonessential restriction site, thereby eliminating the site and allowing selection based on resistance to restriction digestion. No subcloning is necessary with this method, provided that both mutagenic primers are designed to bind to the same strand and that the plasmid contains at least one unique restriction site. The digested mixture of circular mutant and linearized wild-type DNA is used to transfect a mutS strain of E. coli; the mutS mutation renders the cells deficient in mismatch repair, stabilizing the wild-type/mutant heteroduplex. Because the cells are more efficiently transformed by circular DNA, transformation with the mutant is favored. DNA is then isolated from the transformed cells and a second round of selection is performed. Madison, Wis.-based Promega offers the GeneEditor(tm) in vitro Site-Directed Mutagenesis System, promising mutagenesis efficiencies approaching 100 percent by positively selecting for mutants with the company's GeneEditor Antibiotic Selection Mix. The system is based on modification of the ubiquitous TEM-1 ß-lactamase ampicillin resistance (Ampr) gene. After alkaline denaturation of the template DNA, mutant and selection oligos are annealed and the mutant strand synthesized with T4 DNA polymerase. The reaction mixture is used to transform mutS cells, and positives are selected using ampicillin and the GeneEditor Antibiotic Selection Mix. Only the cells containing the modified Ampr gene are resistant to both ampicillin and the GeneEditor antibiotics. Promega's AlteredSites(r) II in vitro Mutagenesis Systems use the pALTER(r) vectors, each of which contains genes for two types of antibiotic resistance, only one of which is functional. In each mutagenesis reaction, the functionality of the resistance genes is reversed using a pair of "on/off" oligonucleotides. Thus, multiple rounds of mutagenesis are possible by switching the two resistance genes on and off. The mutant strand is synthesized with T4 DNA polymerase and propagated in a mutS strain. Finally, PanVera offers the Mutan-Express Km System, manufactured by Takara Shuzo Co. of Tokyo. This system, based on the Oligonucleotide-Directed Dual Amber (ODA) method, uses vectors (pKF18k/19k) that contain two amber (TAG stop codon) mutations in the kanamycin resistance (Kmr) gene. The mutagenic primer and the repair primer are annealed and a complementary strand is synthesized by T4 DNA polymerase. The resulting heteroduplex is introduced into supE/mutS bacteria. When they encounter the amber codon, bacteria harboring a supE mutation will occasionally insert an amino acid instead of terminating translation, thus conferring kanamycin resistance; however, these plasmids will not confer kanamycin resistance in normal (supo) strains. Mutants are therefore selected by re-transformation of clones into a wild-type strain and growth on kanamycin. PanVera's Mutan-Super Express Km kit employs essentially the same approach using patented LA (Long and Accurate) PCR technology, but this kit requires only a single transformation step. Mutants Amplified PCR is a popular way to create site-specific mutations, insertions, and deletions. The technique is fast and can be used with almost any template. Unfortunately, PCR-based techniques also pose technical problems for the researcher. Because most thermostable polymerases have no proofreading capability, there is always the possibility of unintended secondary mutation. In addition, the generation of long PCR products is relatively inefficient. The ExSite(tm) system from Stratagene addresses some of these concerns. According to Stratagene's Alicia Pierini, the ExSite system reduces unwanted second site mutations by increasing template concentration, reducing cycle number, and by using a proprietary polymerase formulation that has a higher fidelity than Taq polymerase. Mutagenesis frequency is improved by removing wild-type, parental DNA through DpnI digestion. The Tfu Direct(tm) kit from Qbiogene of Carlsbad, Calif., also uses a low cycle number to reduce unintentional mutations, and a DpnI digestion to remove parental DNA. This kit employs Qbiogene's high-fidelity Tfu polymerase. PanVera's LA PCR in vitro mutagenesis kit also uses a high-fidelity polymerase, LA Taq(tm), to decrease unwanted mutation. This kit uses PanVera's LA PCR technology to amplify the entire plasmid in two pieces: a 5' fragment, containing the desired mutation at it's 3' end, and an overlapping 3' fragment, extending from the polylinker, through the insert, and back around to the polylinker. The 5' amplification primer for the 3' fragment contains a mutation that will knock out a restriction site in the standard pUC-derived polylinker sequence. By hybridizing the two fragments, extending, and cutting with the appropriate restriction enzyme, the user selects for mutation-containing fragments before recloning. If random mutagenesis, rather than fidelity, is desired, two kits to consider are CLONTECH's Diversify(tm) PCR Random Mutagenesis Kit and Stratagene's GeneMorph(tm) PCR mutagenesis kit. The Diversify kit modulates the mutagenesis frequency during amplification by varying the manganese and dGTP concentration in the reaction, whereas the GeneMorph kit uses an error-prone Mutazyme(tm) DNA polymerase that produces a different mutational spectrum than do Taq-based methods. Potent Transposables If random insertion mutagenesis is required, consider transposon-based mutagenesis strategies. Transposons are DNA sequences that are flanked by specific recombination elements and can "jump" from place to place by the action of a transposase. Any sequence can be randomly inserted into a target if transposase-recognition sites flank it. This type of procedure is particularly useful for screening purposes (e.g., to scan for critical gene sequences, protein-binding sites, or protein domains) because only a single transposable element will insert in any plasmid. The result of the transposition reaction is a pool of targets containing the transposon inserted at random locations. New England Biolab's GPS-LS system can be used for linker-scan analysis New England Biolabs (NEB) of Beverly, Mass., offers two kits based on transposon Tn7. The GPS(tm)-M kit can be used to randomly insert either a kanamycin-resistance cassette, or any other desired sequence, into the target sequence. Because the transposon may be modified, it can be used in a variety of organisms for numerous applications. Unreacted donor molecules can be selectively destroyed by digestion with the restriction enzyme PI-SceI (VDE). NEB's other offering is the GPS-LS kit, which is useful for linker scan analysis of the target DNA. The transposon donor in this system contains a pair of rare PmeI sites that are used to remove the bulk of the transposon after insertion, leaving a short 15-bp sequence. In four of the six possible reading frames, this will result in a five amino acid insertion; in the other two, a stop codon is inserted. Thus, this kit enables testing of both critical DNA elements, as well as protein sequences. Because the donor plasmid in the LS system does not contain a standard origin of replication, unreacted donor is not recovered. The MGS(tm) Mutation Generation System, from Finnzymes of Espoo, Finland (distributed in the United States by MJ Research of Waltham, Mass.), employs a similar linker scanning strategy. This system uses the highly random MuA transposon, which is effective even in GC-rich regions. The bulk of the transposon is excised via NotI digestion, and the resulting 15-bp insertion can then be mapped using a PCR miniprimer. Finnzymes' pEntranceposon plasmids, which enable customization of the transposons, are compatible with this system. Epicentre Technologies, of Madison, Wis., bases its line of EZ::TN(tm) reagents on a "hyperactive" Tn5 transposase. According to Jim Pease, Epicentre marketing manager, Tn5 was chosen because it "is the most random system, which means that [the] transposon will be distributed throughout the target DNA to give insertions into all regions and all sites of the DNA." Epicentre has designed its product line to facilitate in vitro insertions and deletions into cloned DNA, and in vivo insertions into the genomic DNA of living cells. In vitro insertions can be carried out to insert an entire transposon to disrupt gene function or to introduce random, in-frame, 19-codon insertions into cloned genes and cDNA for protein structure/function studies. In vivo mutagenesis is carried out using the EZ::TN Transposome(tm), a preformed DNA-transposase complex so stable that it may be delivered into living cells. Upon internalization of the complex into the cells, intracellular magnesium ions activate the transposase, causing random insertion of the transposon into the host's genomic DNA. Thus, this kit may be used to make random genomic mutations and gene knockouts.4 The Wild Mutant Roundup A variety of other mutagenesis protocols are available. Promoter and protein-function analyses can be aided by the Erase-a-Base(r) system from Promega,5 which uses timed exonuclease III digestion to create a series of nested deletions proceeding from a fixed site on the DNA. The effect of different amino acid substitutions at a specific site within a protein sequence can be ascertained using Promega's Interchange(tm) in vivo Amber Suppression Mutagenesis System. This system includes 13 E. coli strains, 12 of which contain an amber suppressor tRNA that occasionally causes a specific amino acid to be inserted at the site of an amber stop codon; a strain that lacks amber suppression is used as a control. Jeffrey M. Perkel can be contacted at jperkel@the-scientist.com; Aileen Constans can be contacted at aconstans@the-scientist.com. References 1. T.A. Kunkel, "Rapid and efficient site-specific mutagenesis without phenotypic selection," Proceedings of the National Academy of Sciences, 82:488-92, 1985. 2. T.A. Kunkel et al., "Rapid and efficient site-specific mutagenesis without phenotypic selection," Methods in Enzymology, 154:367, 1987. 3. W.P. Deng, J.A. Nickoloff, "Site-directed mutagenesis of virtually any plasmid by eliminating a unique site," Analytical Biochemistry, 200:81-8, 1992. 4. A. Constans, "Transposable Elegance," The Scientist, 14[5]:24, March 6, 2000. 5. S. Henikoff, "Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing," Gene, 28:351-9, 1984.

Mutants Made Easy

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