Setting Your Sites On Genes: Site-directed mutagenesis kits provide fast and efficient peeks into gene function

Date: October 13, 1997 Comparison Chart Knowing the sequence of a gene will never reveal all its secrets. There needs to be a way to tie the sequence of the DNA to the function of the protein. Almost as old as the field of genetics itself is the study of mutations-heritable changes in DNA-through which scientists have attempted to make this connection. With advances in molecular biology technology and in particular the development of the means for synthesizing DNA, mutations are being produced with speed and precision probably never imagined by H.J. Muller and other pioneers in the field of mutagenesis. While there are a number of approaches to in vitro mutagenesis-linker scanning, linker insertions, or nested deletions, to name a few-by far the most common in use today is site-directed mutagenesis and, in particular, oligonucleotide-directed mutagenesis, as revealed by the 12 kits that are in the market today. Oligo-directed mutagenesis uses a mutagenic oligo-usually user-supplied-which, after hybridization to a gene on a plasmid, provides a template for heteroduplex production. The heteroduplexes, containing one normal and one mutant strand, are used to transform bacteria directly in most schemes. During growth of the bacteria, the heteroduplexes are resolved by semiconservative replication into totally mutant and totally nonmutant forms. The trick, and there are almost as many tricks as kits, is to devise a way to isolate efficiently the clones carrying the mutant form of the plasmid. Absent any chicanery, the best one can hope for is 50% recovery of mutants (which is to say that 50% of the resulting clones from bacteria transformed with a heteroduplex, will be mutant), and in actual fact, a good deal lower rates of recovery of mutants are common. With the various techniques in use in mutagenesis kits, efficiencies from 50-100% are reported. The individual techniques employed in the various kits will be described later, but in general, they allow either for negative selection against the parental, nonmutated plasmid or positive selection for the mutated plasmid, both of which significantly raise the efficiency of mutant isolation. Most of the oligo-directed mutagenesis kits allow for variety of types of mutations-point mutations, deletions or insertions. Also, most of the kits do not require subcloning, though a second round of transformation is advised in some cases to improve efficiency. Lastly, as in most areas of molecular biology research, PCR has found an application in the mutagenesis field. While early attempts to incorporate PCR in this field resulted in the introduction of extraneous mutations due to the lack of proofreading activity of most Taq polymerases, several companies have found a way around this problem. In this section, 12 oligonucleotide-directed mutagenesis kits from 9 companies are described. Many are unique in their approach, a marvel of ingenuity. Amersham Sculptor IVM Mutagenesis Kit Based on the method of Fritz Eckstein-modified to improve efficiency-the new Sculptor IVM Mutagenesis Kit from Amersham relies on the inability of certain restriction enzymes to cut a DNA strand at a thionucleotide position. After annealing of user-supplied oligos bearing the desired alteration, extension with T7 polymerase is conducted in the presence of dCTP-S. The resulting heteroduplex is nicked in the non-thio containing, nonmutated strand with an enzyme that is sensitive to the presence of thionucleotides (like Nci I). Following digestion with exo III to create gaps in the nicked, nonmutated strand, T5 exonuclease is brought in to remove the gapped strand. Mutant homoduplexes are made in vitro by final polymerization with pol I and DNA ligase. This can be done in one day and in one tube and requires no special vectors or host cells. Replacing Klenow with T7 polymerase in the initial extension reaction increases the rate of the reaction and the ability to read through difficult regions. In addition, this enzyme avoids loss of mutation due to displacement by newly synthesized DNA. In addition, by using T5 exonuclease to remove unwanted single-stranded templates, template requirement is reduced from 5 to 2 µg of DNA. The kit comes with all necessary enzymes and buffers, dNTP mixtures, host cells, and a control mutant oligo and template. Bio-Rad Muta-Gene In Vitro Mutagenesis Kits Bio-Rad's Muta-Gene Mutagenesis Kit is based on the method of Kunkel, which exploits the fact that uracil-substituted DNA can be selected against in certain strains of bacteria. Uracil-substituted template can be prepared by growing vector in a strain of bacteria that carries two mutations in uracil metabolism, dut- and ung-, which together result in the substitution of uracil for thymine. When such a template is used in the mutagenesis process, and transformed into an ung+ strain of bacteria, the parental, uracil-containing strand is not replicated, leading to the efficient production of mutant containing plasmid. This kit is available either with a single-stranded vector (M13) or a double-stranded phagemid vector. The original version of the kit contains T4 polymerase and gene 32 protein. A newer version of the kit is available with T7 polymerase, which provides a more rapid rate of polymerization and is less likely to terminate prematurely at a region of secondary structure. Included in the kits are enzymes, buffers, host strains and a set of control template and primers. CLONTECH TransformerTM Site-Directed Mutagenesis Kit Transformer Mutageneis Kit is based on the unique site elimination method (USE) of Deng and Nickoloff. Specific mutations are introduced into a target gene cloned into any double-stranded plasmid with a unique restriction site and a bacterial selection marker. Two primers are required: one to introduce the desired mutation and one to mutate the unique restriction site in the plasmid to a different restriction enzyme or to eliminate the unique site. Elongation by T4 DNA polymerase results in the incorporation of both mutations in the same newly synthesized strand. The double-stranded plasmid is digested with a restriction enzyme corresponding to the original unique site, removing all nonmutated plasmid. The DNA is then used to transform E. coli BMH 71-18 mutS, which is repair deficient and will propagate the mutated plasmids. A second round of transformation after redigestion of isolated DNA leads to a very high frequency of mutation in the final transformation reaction. The kit comes with the host cells, enzymes and buffers, and control template and primers. 5 Prime -> 3 Prime Morph Site-Directed Mutagenesis Kit The Morph Site-Directed Mutagenesis Kit relies on the resistance of hemi-methylated but not fully methylated DNA to digestion by the restriction enzyme, Dpn I. Virtually any double-stranded plasmid DNA containing the target sequence of interest can be specifically mutated without use of vector-specific selection or modification oligonucleotides. Target template is propagated in a dam+ bacterial strain, purified and then annealed with one or more user-supplied oligos bearing the mutation(s). Elongation from the oligo(s) by T4 DNA polymerase in vitro results in hemi-methylated, double-stranded, half-mutant plasmids that resist digestion with Dpn I whereas the fully methylated, non-mutant plasmids succumb to digestion. Transformation into repair deficient (mutS) bacteria yields up to 80% mutant plasmid-containing colonies. The kit comes with all enzymes, reagents and competent MORPH mutS cells sufficient for 30 mutagenesis reactions, including five control reactions. PanVera Mutan-Super Express-Km Kit PanVera's Mutan Super Express Km Kit uses PCR amplification and the oligo-directed, dual-amber method for isolating mutants. In this procedure, the vector pKF 18k/19k, which has dual mutations in a kanamycin-resistance gene is grown up in a supE strain of bacteria, the only strain that will support the growth of this vector. After cloning the target gene into this vector, mutagenesis of the target and reversion of the amber mutations in the kan gene are performed simultaneously using a mutagenic oligonucleotide and kan selection primer. E. coli that are sup0 are transformed with the mutated plasmid, and selection for kanamycin resistance is performed. Pharmacia Biotech Unique Site Elimination (USE) Mutagenesis Kit Using the unique site elimination method of Deng and Nickoloff described earlier, Pharmacia's USE Mutagenesis Kit comes with FPLCpure T4 DNA polymerase and FPLCpure T4 DNA ligase, along with preformulated reactions and nucleotide mixes. Two sets of USE Selection/Toggle Primers are available separately, which can be used on most pUC or pBR-derived plasmids. One primer converts the Sca I site in the ampicillin-resistance gene to a Mlu I site. Another selection primer modifies the Ssp I site into a Stu I site. Neither mutation affects ampicillin resistance or plasmid function. Corresponding toggle primers can be used to reinstate the original sites and enable successive rounds of mutagenesis. Also available separately are pGEX USE primers that allow the rapid mutagenesis of DNA inserts that are cloned into any of the pGEX series of bacterial expression vectors. When used with USE Mutagenesis Kit, the primer sets enable a series of site-specific mutations to be successively incorporated into cloned gene sequences without subcloning. Promega Altered Sites II In Vitro Mutagenesis Systems Altered Sites II Mutagenesis Systems introduce mutations into DNA using a dual primer system to simultaneously create site-directed mutations and antibiotic resistance. There are three systems that differ in the vector: pALTER-1 is the original vector; pALTER-Ex-1, and Ex-2, which contain translation initiation codons and ribosome binding sites, allow in vivo and in vitro expression of mutated proteins. The pALTER and pALTER-1 are ampicillin-sensitive; the ampicillin repair oligonucleotide provided is used to convert the vectors to ampicillin resistance in the same reaction in which the cloned insert is mutated. The pALTER-Ex-2 is chloramphenicol sensitive, and is converted to chloramphenicol resistance during the mutagenesis reaction. Screening for antibiotic resistance is a positive selection for the mutant strand. Altered Sites II Mammalian In Vitro Mutagenesis Systems The Altered Sites Mammalian Mutagenesis system uses the same principle as the Altered Sites Directed Mutagenesis System with a mammalian expression/mutagenesis vector. This vector is based on pCI-Neo and is both ampicillin sensitive and chloramphenicol resistant. A mutagenic oligo is used to convert the plasmid to ampicillin-resistance in the same reaction in which the cloned insert is mutated, supplying a positive selection for mutated plasmid. GeneEditor In Vitro Site-Directed Mutagensis System The GeneEditor in vitro Site-Directed Mutagenesis System allows for mutagenesis by coupling selection for the desired mutation to the positive selection for a new antibiotic resistance. Any vector with an ampicillin-resistance gene can be used in this system. Positive selection for mutants occurs by altering the ampicillin-resistance gene in a researcher's vector to confer resistance to the GeneEditor Antibiotic Selection Mix. Synthesis of the mutant strand links the desired mutagenic oligonucleotides and the provided antibiotic-resistance selection oligonucleotide. Only mutants grow, minimizing the screening required. The GeneEditor System can be used for large insertions or deletions and multiple, simultaneous mutations. All reagents required are provided, including competent cells. Quantum Biotechnologies Quant-Essential Site Directed Mutagenesis Kits The Quant-Essential Kit is based on a patented method licensed from the Nation Research Council of Canada involving a closing oligonucleotide that is used in conjunction with a user-specified mutagenic oligonucleotide. Following linearization within the ampicillin resistance gene of uracilated plasmid, the mutagenic primer is annealed to the template The closing oligonucleotide is used to recircularize the same strand of the template, and elongation by T7 DNA polymerase results in incorporation of both primers into the same non-uracilated strand. The completed double-stranded plasmid is transformed into ung+ bacteria, resulting in dual selection. The first selection is based on the fact that only plasmids that have been recircularized with the closing oligo and extended by T7 DNA polymerase will transform bacteria. The second selection is based on the previously mentioned method of Kunkel, which exploits the fact that the mutagenized non-uracilated strands will be preferentially replicated in ung+ bacteria. Insertions, deletions and point mutations can be generated. The kit comes with dut, ung and dut+ bacterial strains, positive control plasmid and primer, and sufficient T7 polymerase to perform 5 or 20 assays. Stratagene QuikChange Site-Directed Mutagenesis Kit ExSite PCR-Based Site-Directed Mutagenesis Kit Chameleon Double-Stranded Site-Directed Mutagenesis Kit The QuikChange Mutagenesis Kit uses double-stranded DNA and two synthetic oligonucleotide primers containing the desired mutation. The oligo primers, each complementary to opposite strands of the vector are extended during temperature cycling by Pfu DNA polymerase. Temperature cycling generates copies of the plasmid by linear amplification, incorporating the mutation of interest. Primers are designed so that they can only extend on the parental strands, disallowing re-amplification and possible error generation in the mutant strand. Digestion with Dpn I removes all pure parental DNA templates (DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to digestion by Dpn I cleavage). The kit comes with two enzymes, Pfu DNA polymerase and Dpn I, competent XL-2 Blue cells, as well as pWhitescript plasmid and two mutagenic primers that restore the beta-gal gene, for a positive control. ExSite PCR-Based Site-Directed Mutagenesis Kit protocol calls for PCR with a set of mutagenic primers, followed by digestion of the heteroduplex with Dpn I to eliminate in vivo methylated parental templates. Using increased template concentrations keeps the cycle number down to less than 10. With appropriate primers, deletions and insertions as well as point mutations can be introduced into genes on plasmids. The kit comes with TaqPlus Long PCR system, cloned Pfu DNA polymerase, Dpn I and control oligonucleotides. The Chameleon Double-Stranded Site-Directed Mutagenesis Kit applies a modification of the unique-site elimination method in which mutagenic primers are used to alter restriction sites within antibiotic resistance genes. This allows for the selective digestion of the nonmutated plasmid. Switch primers are also provided to allow a second round of mutagenesis to recreate the original unique restriction site. The kit comes with selection primers, mutageneic primers for pBluescript II phagemid-competent cells, and enzymes and buffers. Not all site-directed mutagenesis kits are oligonucleotide based. These last two kits offer an alternative to oligo-directed mutagenesis. New England BioLabs Code 20TM Kit 0 Code 20 Kit provides a means of inserting any one of the 20 amino-acid codons at specific sites in DNA using universal mutagenic cassettes. A target molecule lacking a Sap I site must be constructed with a blunt, double-stranded break at the site for mutagenesis. Double-stranded mutagenic codon cassettes, which are three-base pair, direct-terminal repeats with two head-to-head recognition sites for Sap I, are inserted at the target site by ligation. This plasmid molecule containing the cassette is then digested with Sap I, thereby removing most of the cassette, except for a three-base, cohesive extension that is then ligated to create the final insertion or substitution mutation. Each kit comes with 11 double-stranded cassettes, pLITMUS 28, a polylinker vector with no Sap I restriction sites, Sap I enzyme and buffer and enough reagents for 20 amino acid substitutions at 10 different positions. This method does not require a mutagenic oligo, but does require that there be an appropriately positioned restriction site in the gene of interest. Promega Interchange In Vivo Amber suppression Mutagenesis System This kit is a collection of 12 amber suppressor-containing E. coli, with which it is possible to insert one of 12 amino acids at the site of an amber-stop codon in a gene sequence. This approach allows for the study of the effect of up to 12 amino acids in the same site; in experiments with T4 lysozyme, for example, 2015 amino acid substitutions were obtained in 163 positions. Five of the suppressors are derived from chromosomal mutations and the remainder are synthetic constructs on ColE1 plasmids. Any vector can be used with the chromosomal-based suppressors, but pACYC184, supplied with the kit, or others containing an alternate origin of replication, must be used with the plasmid-based suppressors. The strains also contain a chromosomal amber mutation in the arginine biosynthetic pathway, which allows selection for the suppression activity by plating on minimal medium. All the E. coli strains are supplied as competent cells with an efficiency of at least 103 cfu/ug DNA. The following companies list mutagenesis as one of the services they provide; Ana-Gen Technologies Inc., Lofstrand Labs Ltd, Biotech Research Laboratories, Midland Certified Reagent Co, Boston Biomedica Inc., NBI/Genovus, Commonwealth Biotechnologies Inc., Paragon Biotech Inc, Genpak Ltd, PerImmune Inc., Lexicon Genetics Inc. Laura DeFrancesco can be contacted at ldefrancesco@the-scientist.com Comparison Chart

Setting Your Sites On Genes: Site-directed mutagenesis kits provide fast and efficient peeks into gene function

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