BMC Genomics 2007, 8:110 http://www.biomedcentral.com/1471-2164/8/110 libraries of AluI/DraI or RsaI fragments thus constructed were screened by sequencing of plasmid DNA extracted from individual clones using M13 forward and reverse vector primers (Lark Technologies). BLAST searches at the C. jejuni NCTC11168 genome project web site [39] were used to determine the presence or absence of sequences in the NCTC11168 genome. Sequences absent from the genome of C. jejuni NCTC11168 were further analysed using BLASTN and BLASTX searches of the general data- base using the NCBI website [40] (last accessed 13th Octo- ber 2006). PCR amplification and dot-blot screening of strains Oligonucleotide primers (Sigma-Genosys) for PCR ampli- fications are listed in Table 5 along with the annealing temperatures used. DNA for PCR amplifications and dot- blots was prepared by using the Wizard Genomic DNA Purification kit (Promega). For PCR amplification, typi- cally, 1 µl of this DNA was used directly in 25 µl volumes containing 1.25 U of Taq DNA polymerase (Promega), 1 × TaqMaster (Helena Biosciences), 300 nM each primer, 1 × Taq buffer, 2.5 mM MgCl2 and 100 µM nucleotides (dATP, dCTP, dGTP, dTTP). Amplifications were carried out in an Eppendorf MasterCycler thermal cycler for 30 cycles consisting of 95°C (1 min), annealing temperature (1 min) and 72°C (2 min) with an additional extension time at 72°C (10 min) following completion of the 30 cycles. Dot blot hybridisation of genomic DNA was carried out as described previously [38]. Digoxigenin-11-2'-dUTP (DIG) (Roche)-labelled probes were made by carrying out PCR amplification in the presence of 60 µM DIG using vector or internal primers. Hybridisation and subsequent detec- tion of DIG was carried out following the manufacturer's instructions (Roche). All SSH sequence distributions were determined by dot- blot analysis, with the exception of 629-C10, for which a combination of PCR screening and Southern blots was used. This was because background hybridisation made the 629-C10 dot-blots difficult to interpret. A probe for flaA was used in dot blots to confirm the presence of DNA for each strain tested. Authors' contributions PH carried out most of the laboratory work and partici- pated in data analysis. HL carried out the MLST typing. CAH participated in the design, supervision and coordina- tion of the study. CW participated in the design, supervi- sion and coordination of the study, and drafted the manuscript. All authors read and approved the final man- uscript. Additional material Additional File 1 Supplementary Table 1. The table presents a summary of all the SSH sequences obtained in this study. GC (%); percentage G+C content; %ID; % protein sequence identity. 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