Abstract
Nonviral delivery vectors are highly sought after for gene therapeutic applications and genetic vaccination. Dumbbell-shaped DNA minimal vectors have important advantages as compared with plasmids and minicircle DNA. Here, we describe the rapid, cheap, and efficient production of superior dumbbell vectors at high purity using a process termed 1-2-3 gap–primer PCR. This process represents a 1-tube, 2-enzyme, 3 h procedure that comprises a PCR followed by a ligation. The resulting dumbbells harbor mismatches close to the loop structures, which facilitate nuclear diffusion and result in enhanced gene expression.
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Acknowledgments
This work was supported by the National University of Singapore Bridging Grants [NUHSRO/2015/091/Bridging/02 and NUHSRO/2017/068/Bridging/06], the National Medical Research Council of Singapore [New Investigator Grant number NMRC/NIG/1058/2011], and the Ministry of Education of Singapore [Academic Research Fund (AcRF) Tier 1 Faculty Research Committee (FRC) grants number T1-2011Sep-04 and T1-2014Apr-02, the Seed Fund for Basic Science Research number T1-BSRG 2015-05], and the Technology Development Review (TDR) grant number H19H0G1006 of the Agency for Science, Technology and Research (A*STAR) of Singapore all to V.P.
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Loh, P.S., Patzel, V. (2022). Efficient Generation of Superior Dumbbell-Shaped Nonviral DNA Delivery Vectors Using 1-2-3 Gap-Primer PCR. In: Walther, W. (eds) Gene Therapy of Cancer. Methods in Molecular Biology, vol 2521. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2441-8_18
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DOI: https://doi.org/10.1007/978-1-0716-2441-8_18
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