Performance Characteristics

The performance requirements for a good molecular typing system include a high index of strain discrimination (>0.95) and assay reproducibility, and the ability to provide results for a diverse group of microorganisms.[10] Pulsed field gel electrophoresis technology satisfies these criteria and therefore is the most frequently used molecular typing method in molecular epidemiology. The PFGE band patterns represent the entire microbial chromosome profile (>90%).[11] All manipulations for DNA extraction and restriction digestion are performed inside the agarose plug, which protects DNA from shearing forces during sample preparation. Pulsed field gel electrophoresis typically yields 10 to 30 well-separated, easy-to-read chromosomal DNA bands after digestion with infrequent-cutting restriction endonu-cleases. It has significant advantages compared with conventional electrophoresis techniques for analysis of restriction endonuclease digested chromosomal DNA. The latter when applied to conventional electrophoresis cannot separate large fragments, and treatment with frequent-cutting restriction endonucleases will generate hundreds of uninterpretable bands. Therefore conventional electro-phoresis is best suited to the analysis of relatively small-molecular weight (<50 kb) DNA fragments of plasmids. The comparison of the procedural features of conventional electrophoresis and pulsed field gel electrophoresis is summarized in Table 1.

The major disadvantages of PFGE compared to other DNA-based typing methods include the relatively high cost and the labor-intensive techniques that can take 4 to 5 days to complete.[12] DNA fragments smaller than 50 kb cannot be reliably separated by PFGE, because the system is not able to switch the field orientation quickly enough to separate these smaller molecules. Furthermore, certain organisms such as Clostridium difficile and Aspergillus spp. may not be typeable by PFGE as their DNA cannot be isolated intact. Strain differentiation may not be possible for some bacterial species because of inherent nuclease contamination that can degrade DNA.[13]

Many different protocols have been developed in laboratories using PFGE, leading to variability in assay design and reproducibility. It is important to standardize the PFGE protocols, especially, some critical elements, such as the concentration and the integrity of the DNA in the plug, the effectiveness of specific restriction enzymes for digestion, and the electrophoresis conditions including gel volume and agarose concentration, ionic strength and volume of the buffer, and running conditions including voltage, switching times, reorientation angle, and total run times of electrophoresis.1-14,15-1 To insure high-quality gels and consistent reproducibility, we recommend including a quality-control strain with each run for gel comparison.

Table 1 Comparison of the procedural features of conventional electrophoresis and PFGE

Procedural characteristics Conventional gel electrophoresis PFGE

Table 1 Comparison of the procedural features of conventional electrophoresis and PFGE

Procedural characteristics Conventional gel electrophoresis PFGE

Sample preparation

No specific requirement

Intact cells embedded in agarose

Restriction endonuclease

Frequent-cutting enzyme

Infrequent-cutting enzyme

Electric field

Single homogeneous

Pulsed homogeneous

Resolution in DNA size

<50 kb

50-2,000 kb

Typical run times

1-4 hr

18-48 hr

Instrument cost

<$2,000

>$15,000

Interpretation of strain relatedness can be performed by visual inspection of the PFGE bands. Criteria for PFGE interpretation have been proposed for epidemiological investigations1-16-1 in the following manner: bacterial isolates yielding the same PFGE pattern are considered ''indistinguishable;'' isolates differing by one to three bands (a single genetic change) are considered ''closely related;'' isolates differing by four to six bands (two independent genetic changes) are considered ''possibly related.'' Six or more band differences represent three or more genetic changes and such isolates are considered ''unrelated.'' More recently, various computer-assisted programs have become available to enhance the capability of comparing DNA fragment patterns present on multiple gel sets. Using this approach, investigators can create a searchable database of PFGE fragment patterns and easily perform cluster analyses.[17]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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