earticle

영어

Site-directed mutagenesis (SDM) is one of the common methods used in studying the structure-function relationships of proteins. Mutations at the level of nucleic acids are related to altered properties such as the level of expression, the stability, and/or the change in the catalytic activity of a target protein. To this end, nucleotide-directed mutagenesis methods based on polymerase chain reaction (PCR) is widely used. Even though mutagenesis methods are efficient, the accompanying steps such as transformation, colony peaking, and in vivo expression of the mutant proteins are very time consuming. Thus, we integrated the PCR-based
mutagenesis method in the up-stream steps of cell-free protein synthesis to build an efficient system to carry out scanning mutations in parallel. The mutagenic system is devised to enrich mutated DNA while completely removing the residual wild-type template DNA using combination of DNA ligase, polynucleotide kinase, Dpn I, and lambda exonuclease. Results from the proof-of-concept experiment will be presented.