title: Single-step overlap-primer-walk polymerase chain reaction for multiple mutagenesis without overlap extension.
abstract: We present a simple, single-step, single-tube, and rapid method for introducing a series of mutations into cloned DNA. Polymerase chain reaction (PCR)-based mutagenesis methods have become very prevalent due to their simplicity and efficiency for introducing mutations. Our method, overlap-primer-walk PCR, has several advantages over other published methods. It uses two common oligodeoxyribonucleotides and a series of overlapping primers specific for various mutations. Once common flanking primers are selected, two to three mutations require only one additional primer. Therefore, this method is very useful for introduction of multiple mutations in various sites of the target DNA. We illustrate the usefulness of the method by introducing several mutations into the human TNF-alpha encoding gene.
authors: Institute of Microbial Technology, Chandigarh 160036, India.
authors: Raj
authors: R
authors: Kumar
authors: Kammara
authors: K
authors: Rajagopal
mesh_terms: D003365:Costs and Cost Analysis; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D017931:DNA Primers; D014409:Tumor Necrosis Factor-alpha; D004247:DNA