Optimization of overlap extension PCR for efficient transgene construction

Roland S Hilgarth; Thomas M Lanigan

doi:10.1016/j.mex.2019.12.001

Optimization of overlap extension PCR for efficient transgene construction

MethodsX. 2019 Dec 4:7:100759. doi: 10.1016/j.mex.2019.12.001. eCollection 2020.

Authors

Roland S Hilgarth¹, Thomas M Lanigan^{2

1}

Affiliations

¹ Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI, 48109, United States.
² Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, 48109, United States.

Abstract

PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

Keywords: Overlap extension PCR; Restriction enzyme free gene splicing; Subcloning.