129Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_12, © Springer Science+Business Media, LLC 2018
Chapter 12
Direct in Gel Genomic Detection of Antibiotic Resistance
Genes in S1 Pulsed Field Electrophoresis Gels
Mark A. Toleman
Abstract
S1 pulsed field gel electrophoresis (PFGE) is a method to separate the bacterial chromosome(s) from plas-
mid nucleic acids. When combined with ethidium bromide staining and UV visualization this method is
excellent at assessing the number of plasmids in individual bacterial strains. It is also good at approximating
the true size of each individual plasmid when run against a DNA molecular marker. However, downstream
applications such as: the location of individual resistance genes on individual plasmids or the chromosome
are hampered by very poor transfer of large DNA molecules from agarose gels to adsorbent nylon or nitro-
cellulose membranes. Herein, we describe a method to directly probe agarose PFGE gels eliminating the
necessity of transfer and generating excellent genomic location results.
Key words Direct agarose gel probing, Pulsed field gel electrophoresis, Plasmid detection, Genomic
location, Resistance gene mapping1 Introduction
Pulsed field gel electrophoresis (PFGE) is an electrophoresis
method that separates large molecules of DNA in an agarose gel on
the basis of their size [ 1 ]. The parameters of the electrophoresis
module can be altered to separate almost any range of DNA mol-
ecules from small plasmids to whole chromosomes. Large pieces of
DNA such as conjugative plasmids (above 30 kb) and bacterial
chromosomes are typically very fragile and can be sheared by pipet-
ting liquid samples of DNA. For this reason bacterial cultures are
lysed and digested in a solid agarose medium, typically as small
agarose plugs, which are then inserted into an agarose gel. S1 is a
nuclease that primarily has single stranded DNA endonuclease
activity but can also degrade double-stranded DNA at high con-
centration [ 2 ]. Plasmids typically exist in several different struc-
tural conformations mostly based on the degree of supercoiling of
their DNA, many of which do not run at their true molecular size
in agarose gels. Partial S1 digestion nicks plasmid DNA at regions