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Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2967))

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Abstract

The in vivo intramolecular recombination of a parental plasmid allows excising prokaryotic backbone from the eukaryotic cassette of interest, leading to the formation of, respectively, a miniplasmid and a minicircle. Here we describe a real-time PCR protocol suitable for the determination of recombination efficiency of parental plasmids with multimer resolution sites (MRS). The protocol was successfully applied to purified DNA samples obtained from E. coli cultures, allowing a more reproducible determination of recombination efficiency than densitometry analysis of agarose gels.

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Acknowledgement

This work was funded by FCT, Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences, iBB, and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB. Cláudia Alves studies were funded by FCT-Portuguese Foundation for Science and Technology (Grant PD/BD/116842/2016, BIOTECnico Ph.D. program).

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Authors and Affiliations

  1. iBB- Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Cláudia P. A. Alves, Duarte Miguel F. Prazeres & Gabriel A. Monteiro

  2. Associate Laboratory i4HB – Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Cláudia P. A. Alves, Duarte Miguel F. Prazeres & Gabriel A. Monteiro

Authors
  1. Cláudia P. A. Alves
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  2. Duarte Miguel F. Prazeres
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  3. Gabriel A. Monteiro
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Corresponding author

Correspondence to Gabriel A. Monteiro .

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Editors and Affiliations

  1. CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal

    Lucília Domingues

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Alves, C.P.A., Prazeres, D.M.F., Monteiro, G.A. (2023). Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 2967. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3358-8_10

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  • DOI: https://doi.org/10.1007/978-1-0716-3358-8_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3357-1

  • Online ISBN: 978-1-0716-3358-8

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