Method for optimizing pulsed-field gel electrophoresis banding pattern data.
The genomic DNA of 47 strains of TSST-1 toxin-producing Staphylococcus aureus were cleaved with SmaI restriction endonuclease and resolved in an agarose gel by pulsed-field gel electrophoresis (PFGE). An algorithm was designed to standardize the band weights or brightness (trace quantity) produced to a bounded region between 0 and 1 regardless of DNA fragment size while simultaneously reducing gel-to-gel variability. The algorithm allows for classification of isolates by band intensity as well as DNA mobility without a numerical hierarchy of band intensity that is caused by ranging DNA fragment lengths. On analysis many isolates were classified as separate entities on the basis of DNA co-migration only. Isolates differing by only DNA co-migration were subjected to a second digestion with restriction enzyme SacII. These isolates were characterized similarly to the standardized trace quantity analysis of SmaI PFGE patterns. The standardization method proposed in this article permits characterization of isolates on the basis of band differences, regardless of DNA co-migration, thus increasing the discriminatory power (0.79 to 0.89) of PFGE by increasing band-associated information. An established unbiased approach to the partitioning of data were also explored.
Warner
John E
JE
Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. jwarner@rics.bwh.harvard.edu
Onderdonk
Andrew B
AB
D000465:Algorithms; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D025202:Molecular Diagnostic Techniques; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial