Advanced Techniques in Diagnostic Microbiology

148 F. Wu and P. Della-Latta
DNA degradation in the gel. DNA degradation, which is the most common
problem yielding nonspecific fragments ranging from 40 to 150 kb, is due to
the activity of nucleases present in some microorganisms. This problem can be
prevented by the use of HEPES buffer instead of Tris buffer (Koort et al., 2002)
or adding 50–75 μM thiourea in the running buffer to eliminate reactive Tris
radicals (Romling and Tummler, 2000).
Incomplete digestion by restriction endonucleases. On occasion, some enzyme
recognition sites on chromosomal DNA are not cleaved during sample
digestion. This partial digestion results in the production of DNA fragments that
are too large to migrate and therefore remain near the top of the gel. To prevent
enzyme degradation, the protease and detergent added during sample preparation
should be completely removed before adding the restriction enzymes.
Incorrect electrophoresis conditions. To permit microbial genotyping, optimal
electrophoretic conditions for appropriate migration of DNA fragments can be
modeled from previous studies or designed using a standardized marker of known
molecular weight. The use of incorrect settings could cause chromosomal DNA
fragments either to migrate too quickly for retention in the gel or they may be
too close together to interpret.
Analysis of DNA Fragment Length Polymorphism
The DNA fragments in the agarose gel generated by PFGE are visualized by
staining with ethidium bromide. Each lane on the gel represents the chromosomal
pattern of one bacterial isolate. The migration of DNA fragments form patterns
that determine chromosomal similarity and hence clonality of strains. The standardized
recommendations (Tenover et al., 1995) for the interpretation of PFGE
patterns of isolates linked to an epidemiological investigation are dependent upon
the number of band differences on the gel. Those yielding the same pattern should
be considered “indistinguishable,” one to three band differences are “closely related,”
reflecting a single genetic change, four to six band differences are “possibly
related,” representing two independent genetic events, and six or more band differences
represent three or more genetic changes and are considered “unrelated.”
However, comparisons of DNA fragment patterns present on multiple gels from
large sets of isolates are technically difficult to interpret (Chung et al., 2000). There
are variables that might alter fragment patterns and cause lack of interlaboratory
reproducibility, such as type of PFGE instrumentation, protocols, or individual
user techniques. Several commercially available software packages that provide
computerized gel scanning and data analysis can compensate for these intra- and
intergel variations (Duck et al., 2003). For example, the DNA patterns on the same
or multiple gels can be more clearly represented as a dendrogram, showing the percent
similarity obtained through Dice coefficients and the unweighted pair group
method with arithmetic average. Through the use of computer-assisted analysis
of DNA fragment polymorphism, investigators are able to create searchable
databases of DNA patterns for multilaboratory comparison and for future strain
comparisons.