One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections (original) (raw)
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Biotechnology Letters, 2007
Site-directed mutagenesis is of great importance for probing the structure/function relationship of proteins. Developing our previous method (Nagy et al. Anal Biochem 324:301-303, 2004), here we report a multiplex strategy for site-directed mutagenesis using PCR in one tube to introduce a single mutation into three or more genes at the same time. DNA fragments carrying the desired mutation can be distinguished from each other in a standard antibiotic selection step of the transformed bacteria. Due to this strategy the mutagenesis procedure for several genes can be accelerated.
PCR-based strategy for construction of multi-site-saturation mutagenic expression library
Journal of Microbiological Methods, 2007
There is an increasing demand for efficient and effective methods to engineer protein variants for industrial applications, structural biology and drug development. We describe a PCR-based strategy that produces multi-site-saturation mutagenic expression library using a circular plasmid carrying the wild-type gene. This restriction digestion-and ligation-independent method involves three steps: 1) synthesis of the degenerate oligonucleotide primers, 2) incorporation of the mutations through PCR, 3) transformation into the expression host. Our strategy is demonstrated through successful construction of an E. coli K12 malic enzyme expression library that contains members with simultaneous mutations on amino acid residues G311, D345 and G397. This method is in principle compatible with any circular vector that can be propagated with a dam + E. coli host to generate protein variant library with multiple changes, including mutation, short sequence deletion and insertion, or any mix of them.
Rapid and efficient site-specific mutagenesis without phenotypic selection
Methods in enzymology, 1987
Several single-base substitution mutations have been introduced into the lacZa gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a sitespecific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, %10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung') Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
PloS one, 2013
Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature o...
A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence
Biotechnology and Applied Biochemistry, 2008
Background site-specific mutagenesis at one or multiple sites has recently become an invaluable strategy in functional proteomic studies and genetic engineering. In the present paper we describe a novel PCR-based procedure for site-specific mutagenesis that permits, in a single-step, all three types of nucleotide sequence mutation (deletion, insertion and substitution). The entire procedure is carried out in one tube and takes about 3-4 h. The method utilizes two primers, one of which is phosphorylated at the 5 -terminus, that are designed to directly anneal back-to-back to the target sequence inserted in a plasmid. For the deletion type of mutagenesis (which has virtually no limit to its extent), primers anneal at the ends of the sequence to be deleted. For insertion and substitution types of mutagenesis the primers bear the mutagenic sequences in a tail. The entire circular plasmid, here tested for a maximum length of 7 kbp, is amplified by inverse PCR. The PCR product incorporates the desired mutagenesis and, after ligation, the plasmid is ready for cloning into bacteria. The method has been proved very efficient for deletions of up to 279 nucleotides, for introducing simultaneous deletions, insertions and substitutions, and for performing alanine scanning over a wide coding region. The procedure is suitable for applications in genetic engineering and for the construction of libraries.
A novel PCR strategy for high-efficiency, automated site-directed mutagenesis
Nucleic Acids Research, 2005
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 together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T m mutagenic primer at a high annealing temperature, which prevents the priming of the low T m primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated.
Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template
Synthetic Biology
Directed DNA libraries are useful because they focus genetic diversity in the most important regions within a sequence. Ideally, all sequences in such libraries should appear with the same frequency and there should be no significant background from the starting sequence. These properties maximize the number of different sequences that can be screened. Described herein is a method termed SLUPT (Synthesis of Libraries via a dU-containing PCR Template) for generating highly targeted DNA libraries and/or multi-site mutations wherein the altered bases may be widely distributed within a target sequence. This method is highly efficient and modular. Moreover, multiple distinct sites, each with one or more base changes, can be altered in a single reaction. There is very low background from the starting sequence, and SLUPT libraries have similar representation of each base at the positions selected for variation. The SLUPT method utilizes a single stranded dU-containing DNA template that is ...