DNA band patterns were obtained after gel electrophoresis (0.8% agarose gel) of the RAPD-PCR reaction products (15 μL). Gels were run for about 55 min at 100 V and stained in ethidium bromide (0.5 μg mL−1) for 30 min. DNA molecular weight marker (‘500 bp molecular ladder’, Bio-Rad) was used as a standard. Gel
images were processed using the software fingerprinting II (Bio-Rad). The similarity matrix was calculated on the basis of the Pearson product–moment correlation coefficient, and its corresponding dendrogram was deduced using the unweighted pair group method with arithmetic averages [Struelens PI3K inhibitor & the Members of the European Study Group on Epidemiological Markers (ESGEM), of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID), 1996].
Reproducibility was assessed by cluster analysis of triplicate reactions. RAPD-based methods do not require sequence information for PCR primer design. However, they are extremely dependent on laboratory conditions such as template DNA concentration, PCR and electrophoretic settings, among others (Ellsworth et al., 1993). To establish a quick and useful RAPD-PCR protocol to type phages, phages infecting strains belonging to the same species (four Staphylococcus epidermidis phages), or different species within the same genus (two Staphylococcus aureus phages) or a different genus (one Lactococcus lactis phage) were selected to test several experimental conditions in order to generate see more reproducible RAPD patterns and gain a preliminary insight into the discrimination power of this approach. The selected S. epidermidis phages belonged to the Siphoviridae family (morphotype B1) and their genome sequences were unknown. However, previous
DNA restriction analysis revealed distinct patterns for the temperate phages ΦSepi-IPLA6 and ΦSepi-IPLA7, while the DNA restriction patterns of the lytic phages ΦSepi-IPLA4 and ΦSepi-IPLA5 (presumably virulent derivatives of ΦSepi-IPLA6) were very similar to each other (Gutiérrez et al., 2010; and our unpublished data). The two phages infecting 5-FU research buy S. aureusΦH5 and vB_SauS-phiIPLA35 (Φ35) belonged to morphotype B1 and morphotype B2, respectively, and their complete genome sequence was available (García et al., 2007, 2009). Finally, the lytic L. lactis phage ΦC2 belonging to the morphotype B2 (Lubbers et al., 1995) was chosen as representative of phages infecting a different genus within Gram-positive bacteria. Initially, pure phage DNA (10 ng) was used as a template. Because RAPD-PCR reactions are considerably influenced by primers and their concentration (Johansson et al., 1995), four primers (OPL5, RAPD5, P1 and P2) at three different concentrations (1, 4 and 8 μM) were tested. Furthermore, we tested whether the presence of magnesium oxalacetate and DMSO resulted in better defined band patterns.