Strains were grown in a tryptose-peptone-glucose-yeast medium (Difco Laboratories, Detroit, Mich.) under anaerobic conditions at 37°C 16 h.
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted as described by Hyytiä et al. (1999) and Keto-Timonen et al. (2003). In brief, the cells were lysed in TE containing lysozyme and mutanolysin. Lysis was completed by adding proteinase K, NaCl, EDTA, and sodium dodecyl sulfate. Phenol-chloroform-isoamyl alcohol and chloroform-2-pentanol extractions were performed, and DNA was ethanol precipitated and resuspended in TE. RNA was removed by RNase , followed by the addition of NaCl, chloroform-2-pentanol extraction, and ethanol precipitation. DNA concentrations were determined with a BioPhotometer (Eppendorf, Hamburg, Germany).
Label
Cy5
Label protocol
DNA was labeled using the BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. Labeled DNA was purified with the DNA purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany).
Hybridization protocol
Agilent (Santa Clara, USA) 8x15K CGH protocol, 65°C for 18-20 h.
Scan protocol
GenePix 4200 AL (Axon Instruments) using pixel resolution of 5 µm, line average 2.
Data processing
Image analysis with GenePix Pro 6.0, bad spots were manually flagged (flag-value -100). Data preprocessing in R with limma package: background subtraction with normexp and offset 50. Normalization factor was determined by the location of the main mode of log-ratio distribution between the sample and reference strain. Separate normalization factors were calculated between each sample and all reference hybridizations. Normalized log2-intensity was determined by subtracting the sample-specific median normalization factor from the original log2-intensity.
Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A strains
