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

Journal of Biological Engineering
Sayed Shahabuddin Hoseini, Martin G Sauer

Abstract

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. 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....Continue Reading

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Citations

Jul 24, 2020·PloS One·Lorenz Tim SparrenbergHarald Peter Mathis
Sep 24, 2016·Quarterly Reviews of Biophysics·Kathleen A LeamyPhilip C Bevilacqua
May 8, 2018·Applied Microbiology and Biotechnology·Chiqian ZhangZhiqiang Hu
Dec 19, 2020·Current Medical Imaging·Nitin SahaiRavi Prakash Tewari

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Datasets Mentioned

BETA
EF694056.1

Methods Mentioned

BETA
PCR
restriction digest
transfection
fluorescence microscopy
fluorescence
flow cytometry
FCS

Software Mentioned

excel
SciEd
Phusion
Image [UNK]
BLAST
Clone Manager suite
FlowJo
Clone Manager

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