Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.

BACKGROUND: Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning. RESULTS: Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells. CONCLUSIONS: Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.