Sample Preparation for PFGE

Standard sample preparation procedures for DNA isolation involving mechanical forces are inappropriate for the analysis of fragile, high-molecular weight DNA molecules that easily break into small pieces. To prevent DNA damage, intact cells mixed with warmed, liquid agarose are pipetted into plug molds, 10 x 5 x 1.5 mm in size. Cells embedded in the agarose plugs are lysed in situ by detergents and enzymes. The agarose matrix keeps the large DNA molecules intact, while permitting diffusion of lysed cellular components from the plug. Infrequent-cutting restriction endonucleases are used to digest the DNA. The choice of restriction enzyme is critical because it should generate between 10 and 30 numbers of DNA fragments necessary for strain discrimination. A list of endonucleases for typing specific microorganisms have been published.[9] After restriction enzyme digestion, the plugs are cut into appropriate sizes based on the DNA concentration and loaded into the wells of an agarose gel. The gel is then placed in the electrophoresis chamber, and pulsed field conditions are chosen based on the expected size range of the DNA. To separate 40-2000-kb-sized DNA fragments, PFGE is usually run for 18-48 hr. After staining with ethidium bromide, bands are visualized and photographed. Several commercially available software packages are available that provide computerized gel scanning and data analysis capabilities. They enable the clustering pattern of each gel to be represented as a dendrogram that shows the percent similarity obtained through Dice coefficient and the unweighted pair group method with arithmetic average. Pulsed field gel electrophoresis patterns can also be stored for future strain comparisons.

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