CHEF PROCEDURES: A RAPID HIGH-TEMPERATURE METHOD FOR SAMPLE PREPARATION, A HIGH VOLTAGE HEPES BUFFER SYSTEM AND THE USE OF NUSIEVE® AGAROSE

ABSTRACT A rapid, high-temperature method of sample preparation from gram-negative bacteria for contour-clamped homogenous electric field (CHEF) electrophoresis is described, which utilizes a diethylpyrocarbonate nuclease inactivation step for preventing latent degradation of DNA. Also described is a simple 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system that permits high-voltage CHEF electrophoresis in standard apparatus while preventing the strand scission events associated with tris(hydroxymethyl)aminomethane buffers. Finally, the use of NuSieve agarose in combination with standard analytical agarose was compared to specialty CHEF agarose, demonstrating the potential use of this reagent in CHEF gel separations. PRACTICAL APPLICATIONS Contour-clamped homogenous electric field (CHEF) gel electrophoresis is a widely used technique in epidemiological and comparative genetic studies of microorganisms. Standard techniques work variably well, making options that can be used to “tweak” methodology useful. In this article, we describe a rapid, higher-temperature variation of sample preparation that yields inherently stable DNA with a more efficient usage of expensive reagents. Also detailed is a simple electrophoresis buffer that eliminates the tris(hydroxymethyl)aminomethane-mediated strand scission events that have complicated the comparison of bacterial strains in some studies. This buffer permits the use of the higher-voltage electrophoresis conditions used in many studies without the addition of potentially harmful chemicals. Finally, we demonstrate that the combination of NuSieve (Lonza) branded agarose with standard agarose can be used effectively in CHEF gels, thus expanding analytical options while also providing an alternative to maintaining other expensive specialty agaroses. The methods herein may be applied in whole or in part, allowing their possible inclusion in existing or future protocols as desired.