Saturday, November 19, 2011

Restriction Enzyme Pattern in Clinical Microbiology

Restriction endonucleases recognize specific nucleotide sequences inDNA and produce double-stranded cleavages that break the DNAinto small fragments. The number and sizes of the restrictionfragments, called restriction fragment length polymorphisms (RFLPs) , generated by digesting microbial DNA are influenced by both therecognition sequence of the enzyme and the composition of theDNA. In conventional restriction endonuclease analysis, chromosomalor plasmid DNA is extracted from microbial specimens and thendigested with endonucleases into small fragments. These fragmentsare then separated by size with use of agarose gel electrophoresis.The nucleic acid electrophoretic pattern can then be visualizedby ethidium bromide staining and examination under UV light(Lodish et al., 2004).
Restriction endonuclease analysis has the advantage of being highly reproducible, very accurate in determining the relatedness of microbial strains, and well within the technical capabilitiesof experienced laboratory technologists. However, the majorlimitation of this technique, especially for chromosomal DNA,is the difficulty of comparing the complex profiles generated,which consist of hundreds of fragments. To address this problem,pulse-field gel electrophoresis (PFGE) has been developed to enable the separation of large DNA fragments. PFGE providesa chromosomal restriction profile typically composed of 5 to20 distinct, well-resolved fragments ranging from ~10–800kilobases (kb). The relative simplicity of the RFLP profilesgenerated by PFGE facilitates application of the procedure inidentification and epidemiological survey of bacterial pathogens. Fingerprinting,which combines PFGE with Southern transfer and hybridization,has been widely used in studying the tuberculosis nosocomialoutbreak in human immunodeficiency virus (HIV)-positive populations(Goering .,2004).

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