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

Cláudia P A Alves; Duarte Miguel F Prazeres; Gabriel A Monteiro

doi:10.1007/978-1-0716-3358-8_10

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

Methods Mol Biol. 2023:2967:117-131. doi: 10.1007/978-1-0716-3358-8_10.

Authors

Cláudia P A Alves^{1

2}, Duarte Miguel F Prazeres^{1

2}, Gabriel A Monteiro^{3

4}

Affiliations

¹ iBB- Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
² Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
³ iBB- Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. gabmonteiro@tecnico.ulisboa.pt.
⁴ Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. gabmonteiro@tecnico.ulisboa.pt.

PMID: 37608107
DOI: 10.1007/978-1-0716-3358-8_10

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.

Keywords: Intramolecular recombination; Minicircles; ParA resolvase; Real-time PCR; Recombination efficiency.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli* / genetics
Eukaryota*
Eukaryotic Cells
Real-Time Polymerase Chain Reaction
Recombination, Genetic