Advanced Techniques in Diagnostic Microbiology

15
Detection and Characterization of
Molecular Amplification Products:
Agarose Gel Electrophoresis, Southern
Blot Hybridization, Restriction Enzyme
Digest Analysis, and Enzyme-Linked
Immunoassay
RAYMOND P. PODZORSKI,MIKE LOEFFELHOLZ, AND RANDALL T. HAYDEN
Introduction
The need for accurate detection and characterization of nucleic acid targets has
prompted the development of a range of methodologies. Highly complex and often
expensive techniques, such as oligonucleotide arrays, are being used increasingly.
Such methods can be extremely valuable, but issues such as cost, the need for
specialized equipment, and a high level of expertise for both the technical and
analytical aspects of implementation may limit their use in a clinical setting (Chee
et al., 1996; Cheung et al., 1999). Although such systems are certainly effective for
gathering large amounts of information and can be extremely useful in the research
arena (Khan et al., 1999), their use may be unnecessary if only single PCR target
detection is required. The use of real-time molecular product detection methods,
largely relying on the principle of fluorescent resonance energy transfer (Chen et al.,
1997) (FRET), has also become quite commonplace. These methods are useful for
high-throughput diagnostic assays and are amenable to automation.
Some may think that these recently developed techniques have completely supplanted
more traditional procedures, such as agarose gel electrophoresis, Southern
blot, and restriction fragment length polymorphism (RFLP) analysis and even
somewhat newer techniques, such as enzyme immunoassay (EIA). However, such
methods remain useful, often critical tools for research and assay development, and
sometimes still have advantages for clinical testing. All are easy to perform, require
relatively inexpensive equipment, and can be mastered in a short period of time.
Few methods of detecting a PCR product are as easy and inexpensive as pouring
an agarose gel, electrophoresing the PCR product through the gel, and visualizing
the PCR product with a UV light source. The advantages of such direct product
visualization are irreplaceable in assay development and troubleshooting. In cases
of frequent target variation, such methods may offer the only practical means of
positive, reproducible detection and characterization. EIA-based methods remain
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