Advanced Techniques in Diagnostic Microbiology

9. Pulsed-Field Gel Electrophoresis 145
Bacterial Cell Lysis and Release of Intact Chromosomal DNA
A pure culture of a bacterial isolate of known identity is incubated overnight in a
nutrient broth, such as trypticase soy broth (BD Biosciences, Sparks, MD, USA) in
order to achieve 10 9 cells/mL, which is the concentration needed to obtain a visible
band pattern. Standard DNA extraction procedures are inappropriate for the analysis
of large chromosomal DNA molecules because of DNA shearing caused by the
application of mechanical force in the protocol. In order to prevent DNA damage,
it is important to mix intact bacterial cells with warmed, liquid-phase agarose,
and this mixture can then be pipetted into plastic molds to form 10 × 5 × 1.5mm
agarose plugs. The whole cells embedded in plugs are lysed and deproteinized by
detergents and enzymes (e.g., lysostaphin, lysozyme, proteinase K, mutanolysin
or lyticase) in situ (Table 9.1). Following cell lysis, the plugs are washed 4–5 times
with wash buffer containing 20 mM Tris and 50 mM EDTA (pH 8.0) to remove
cell debris and proteinase. The agarose gel matrix keeps chromosomal DNA intact
while removing the rest of cellular components from the plug. This step, including
the post-lysis washing, usually takes 2 days. In recent years, several investigators
have reported improvements to the traditional DNA preparation process. The most
notable time-saving approaches have included (i) directly using bacterial colonies
grown on plates of clinical specimens, (ii) using a combination of lytic enzymes,
(iii) adding lytic enzymes to the bacterial suspensions before preparing the agarose
plugs, (iv) shortening the cell lysis time by reducing the size of agarose plug, and (v)
expediting the wash steps by using a large volume (10 mL) of preheated (50 ◦ C)
water and TE buffer (Gautom, 1997; Turabelidze et al., 2000; Lopez-Canovas
et al., 2003). Of particular interest, a DNA purification system was designed to
automate all steps involved in the preparation of DNA plugs for PFGE (Fiett et al.,
2004).
Restriction Endonuclease Digestion of Chromosomal DNA
A large amount of clean, intact chromosomal DNA embedded in agarose plugs can
be easily digested with a variety of restriction endonucleases. Each of those enzymes
is found to cleave double-stranded DNA at a specific nucleotide sequence,
known as the enzyme’s recognition site. Once the recognition site is located, the
enzyme catalyzes the digestion of DNA at that defined position either close to
or within the targeted sequence, causing a break in the nucleic acid strand, and
producing discrete restriction fragments. The choice of the restriction enzyme is
dependent upon the bacterial species studied. The number and size of fragments
generated by an endonuclease depends on the frequency of the specific restriction
enzyme recognition sites located on a particular bacterial genome. This cutting
frequency is a function of the number of base pairs required for enzyme recognition
(longer sequences lead to less enzyme recognition), and the GC content of
the genome. For example, the restriction enzyme, SmaI, recognizes the CCC/GGG
sequence that cleaves the DNA of most Gram-positive bacteria, whereas XbaI recognizes
the T/CTAGA sequence that cleaves that of many Gram-negative bacteria.