High efficiency of site-directed mutagenesis mediated by a single PCR product
نویسندگان
چکیده
منابع مشابه
Site-directed mutagenesis using PCR-mediated introduction of silent mutations.
The alteration of a specific sequence of DNA using site-directed mutagenesis (SDM) provides an effective tool to probe the structure and function of gene products. With the advent of polymerase chain reaction (PCR), several new techniques were developed that facilitate SDM. An oligonucleotide containing mismatches at the desired site of mutation is used to generate a population of mutant fragme...
متن کاملA novel PCR strategy for high-efficiency, automated site-directed mutagenesis
We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T(ra)), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T(m)) are used togethe...
متن کاملModification of a PCR-based site-directed mutagenesis method.
Site-directed mutagenesis is a powerful tool for producing mutants to assess the importance of specific amino acid residues in a protein’s structure and/or function. We wanted to generate mutants of human ETS1 cDNA in the pET15b vector (Novagen, Madison, WI, USA) from which we had been producing wild-type protein for structural studies (11). Since we were subject to the constraints of this vect...
متن کاملSite-directed mutagenesis of large (13-kb) plasmids in a single-PCR procedure.
Although there are many methods for site-specific mutagenesis, most are only applicable for short (< 7-kb) plasmids. The few methods that have been developed for longer plasmids are complicated procedures that involve multiple PCR steps or complex recombination and ligation reactions (2,4–6). Here, we provide a simple single-PCR procedure for site-specific mutagenesis of long plasmids (13 kb), ...
متن کاملSite-directed, recombination-mediated mutagenesis of a complex gene locus.
We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae. This gene cluster contains four beta-type globin genes which share a high degree of sequence homology. In this procedure, a small fragment of beta A-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integratin...
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ژورنال
عنوان ژورنال: Nucleic Acids Research
سال: 1997
ISSN: 1362-4962
DOI: 10.1093/nar/25.3.682