Advanced Techniques in Diagnostic Microbiology

244 R. P. Podzorski, M. Loeffelholz, and R. T. Hayden
simple, inexpensive to develop and use, rely on commonly available equipment,
and offer excellent analytical performance characteristics. This chapter will discuss
the use of agarose gel electrophoresis, Southern blot hybridization, RFLP analysis,
and PCR-EIA as methods for the detection and characterization of PCR products.
Agarose Gel Electrophoresis
Principles
Agarose is a polysaccharide composed of long chains of cross-linked galactopyranose
residues (Sambrook et al. 2005) substituted with pyruvate, sulfate, and
methyl esters. The pore size of agarose (determined by agarose concentration in
the gel) is responsible for much of its DNA separation properties. DNA molecules
migrate at a rate that is primarily size-dependent. The rate of such movement is inversely
proportional to the log 10 of the length of the DNA strand, such that smaller
molecules of nucleic acid move more quickly than large ones. DNA molecules
of approximately 20,000 bp are the largest molecules that can be resolved using
continuous-field (electrical current) agarose gel electrophoresis. Larger DNA
molecules require methods such as pulsed-field gel electrophoresis (Arshad et al.,
1993; Finney, 2000; Sambrook et al., 2005). One factor that can slow DNA migration
and hinder separation, particularly of larger DNA fragments, involves electroendosmosis
(EEO). EEO is dependent on the number of sulfate and pyruvate
residues present in a given agarose gel (Upcroft and Upcroft, 1993; Sambrook
et al., 2005). It results from positive ions moving toward the cathode, pulling water
molecules with them in opposition to the migration of negatively charged DNA
toward the anode. The effects of EEO are seen mostly in resolution of fragments
>10 kb.
The supplies and equipment needed to perform agarose gel electrophoresis make
it one of the most readily performed, widely available, and inexpensive molecular
methods. Horizontal slab gels, loading and running buffers are often prepared inhouse
but can be purchased from commercial suppliers, with precast gels available
in a range of sizes, using various concentrations of agarose. Hardware required for
agarose gel electrophoresis consists of an electrophoresis gel box and gel casting
tray, gel combs (used to make loading wells or slots), a microwave oven or hot
plate, and an electrophoresis power supply (Sambrook et al., 2005). UV light–
transparent material should be used to make the casting tray so it can be placed
directly on a UV transilluminator. A fitted cover on the gel box is necessary to
prevent contact with running buffer during electrophoresis. Although gel boxes,
casting trays, and combs are relatively simple to construct, commercially available
hardware is widely available.
Technical Considerations: Gel Performance
Factors effecting gel performance and the ability to resolve DNA fragments include
both characteristics of the gel itself (agarose concentration, class, and grade),