Site-directed mutagenesis refers to a man-made molecular biology method that is used to make genetic alterations in the DNA sequence of a gene of interest. But based on our recently published experimental findings, we propose that “natural site-directed mutagenesis” might exist in eukaryotic cells, which is triggered by harmful agents and co-directed by special transcription hotspots and mutati...

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the me...

BACKGROUND Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 1...

background: among all common techniques in sitedirectedmutagenesis, λ red recombinase system has beenwidely used to knock out chromosomal genes in bacteria. in thismethod, there is always the risk of dna linear digestion byhost's restriction enzymes that leads to the low frequency ofrecombination. objectives:to overcome this, we constructeda recombinant vector to disrupt phop gene in salmonella...

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying...

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 st...