查看文章

DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow …