Nonspecific primer and PCR generated hybridization probes for physical ordering large restriction fragments in complex genome of S. aureus

Pulsed field gel electrophoresis (PFGE) allows separation of large restriction fragments from bacterial genome. Restriction fragments obtained by digestion of Staphylococcus aureus DNA with rare cutting enzymes (Sma I, and Csp I) were separated by PFGE. To arrange the physical order of the fragments generated by digestion with one enzyme, probes were prepared by nonspecific priming and polymerase chain reaction (PCR), using individual fragments of the other enzymatic digest as a template. Probes were then used for Southern hybridization to the PFGE separated fragment distribution of the two infrequent cleaving enzymes (Sma I and Csp I). Using probes generated from four Sma I fragments and five Csp I fragments as individual templates, a partial physical order of Csp I fragments of the genome of S. aureus ISP8 has been determined in relation to a previously published Sma I map of S. aureus genome.