Microarray-based Detection of SPI-7 Genetic Elements

A custom oligonucleotide probe-based array was designed as previously described [92] (link) in order to detect genes related to the absence and presence of SPI-7. After labelling, probes were purified and applied to microarray slides [93] (link). Genomic DNA was sonicated to yield 200–500 bp fragments, purified and labelled with Cy3-dCTP using the BioPrime DNA Labelling System (Invitrogen–BioSciences Ltd., Dun Laoghaire, Ireland). Duplicate slides were hybridized with the dCTP labelled DNAs in 48% formamide at 55oC for 16–20 hrs in a humid chamber. The slides were washed at RT, washed again at 50oC, scanned (GenepixR 4000B laser scanner, Axon Instruments, Redwood City, Calif.) and processed (GenePix Pro 3.0). The full dataset was analyzed using R (www.r-project.org), and Bioconductor (www.bioconductor.org) as described [94] (link). In brief, the bimodal distribution that was observed was treated as two overlapping Normal distributions. Means and 95% confidence intervals were determined for each distribution. Probes were scored “absent” if the log2 intensity was within or below the 95% CI for the “low” peak; “present” if the log2 intensity was within or above the 95% CI for the “high” peak and intermediate values were scored as “uncertain”. As a control, PCR tests similar to those described previously [95] (link) were used to screen for presence or absence of larger regions of SPI-7.