Design of synthetic oligonucleotide duplexes for site-directed mutagenesis with recombinant plasmids

Screening for site-mutated plasmids may be greatly facilitated by the different occurrence of the restriction sites distinguishing the altered sequence from the original one. Synthetic oligonucleotides can be so designed that they, apart from defining the mutation, also create a new restriction site recognizable in the restriction pattern of the mutant plasmid.     

An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.     

Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced inEscherichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritins were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.     

A marker-coupled method for site-directed mutagenesis

A marker-coupled method for site-directed mutagenesis (SDM) has been developed. In this method, target DNA is first cloned into a plasmid vector which carries an inactivated tetracycline-resistance (TcR)-encoding tet gene. Using this cloned plasmid as template, polymerase chain reaction (PCR) is performed with a mutagenic primer and a marker primer. The mutagenic primer contains the desired mutations to be introduced into the target DNA, and the marker primer contains a mutation for restoring the activity of the inactivated tet gene. The PCR product is annealed with a gapped duplex plasmid template, extended and ligated in vitro. The resulting uni-strand-mutated plasmid is converted into the gapped duplex form, transformed into Escherichia coli JM109 and spread on yeast extract/tryptone culture medium + Tc plates. The TcR colonies grown on these plates all carry active tet genes. Due to the 'tight coupling' between the marker primer and the mutagenic primer formed in the PCR product, these TcR colonies should also carry the mutagenic primer, e.g., the desired mutations in the target DNA. In fact, practically all of the TcR colonies have been found to be the desired mutants in the present experiments. Therefore, this method provides a very efficient approach for SDM.     

Rapid and economical multiple base site-directed mutagenesis of double stranded plasmid DNA

This method for the mutagenesis of ds-DNA, utilizing the best features of previously published protocols, incorporates a fragmentation procedure on the plasmid, thermostable enzymes and two transformations in E. coli. Screening of positive clones can begin after about two days. Insertions, deletions and substitutions of up to 50 bp are routinely obtained with 90-95% of clones positive as proven by sequencing. The cost is about one half to one third of equiv-alent commercial procedures.     

Primer-directed mutagenesis of linearized plasmids

Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed. In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template. The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA. These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis.     

Saturation site-directed mutagenesis of thymidylate synthase

We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.     

Activation of aspartase by site-directed mutagenesis

To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.     

In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.     

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used.