A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification...

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully ca...

Introduction of multiple mutations can be accomplished through phage M13-based site-directed mutagenesis using several oligonucleotides (6). The major drawback of this method is the low efficiency of generating all the intended mutations simultaneously (7,9). The alternative methods for introducing multisite mutations are based on polymerase chain reaction (PCR) (1) and ligase chain reaction (L...

background: causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. brucellosis is the most common zoonotic infectious disease that would cause great economic losses. thus, recognition of pathogenic and immunogenic factors in the genus brucella can lead to control this health problem. objectives: considering the importanc...

Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein’s structure and function. Objectives: We introduced a nested-SOE-PCR (N –SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.   Materials and Methods: Genomic DNA from Bacillus thermo...