PMC:3091627 / 26641-29905
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3091627","sourcedb":"PMC","sourceid":"3091627","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3091627","text":"In house DNA macroarray membrane preparation\nA total of 254 PCR products were amplified in four 96-well microtiter plates, corresponding to 41 ubiquitous and 213 non-ubiquitous genes from the genome of strain 26695 as previously described [39]. Briefly, amplification reactions were performed in 2 × 100 μl reaction volumes, in which 2 μl of DNA corresponding to the recombinant plasmid containing the full-length CDS (CoDing Sequence) inserted into the pILL570-derivative vector was used as template. Each PCR product was sequenced to confirm the identity of the gene, and was then spotted in triplicate onto a nylon membrane (Qfilter, Genetix 22.2 × 22.2 cm, N+) using a Qpix robot (Genetix). Denaturated 26695 genomic DNA was spotted in triplicate at the four corners of the membrane (positive controls) and seven squares were left empty as negative controls. Following spot deposition, membranes were fixed for 15 minutes in 0.5 M NaOH 1.5 M NaCl, washed briefly in distilled water, and stored wet at -20°C until use [39].\nAliquots of 250 μl of DNA were labeled by random priming with 2 μl of 33P-dCTP. Labeling was performed for 3 hours at room temperature. Unincorporated radionucleotides were removed by purification on Quick Spin Sephadex G-25 columns (Roche Diagnostics). Immediately before being used for hybridization experiments, the sonicated, labeled, and purified chromosomal DNA was heat-denaturated and cooled on ice. Hybridization was conducted in 5 ml prewarmed (65°C) hybridization mixtures containing the heat denaturated probe, with overnight incubation. Membranes were then washed and exposed for 25 hours to a phosphoimager screen (Molecular Dynamics).\nScreens were scanned on a Storm 860 machine (Molecular Dynamics). Image analysis and quantification of hybridization intensities for each spot were performed using the Xdots Reader program (COSE) and determined in pixels [39]. The intensity of the background surrounding each spot was substracted from that of each of the spots. Twenty-one homologous hybridizations were performed. The average intensity of the 41 ubiquitous genes was calculated for each reference array. This number served to allocate a reference array to each heterologous hybridization (average of the ubiquitous spots from the heterologous and the homologous reference hybridizations were not significantly different, Student's test), to calculate the ratio used for normalization. Following normalization, the data were analyzed by attributing a binary score (presence/absence - Additional file 1) or by multidimensional analysis based on continuous intensity values (Figure 1 and Figure 2). To define the cutoff ratio for the presence/absence of a gene, we analyzed the results for the sequenced H. pylori J99 DNA hybridized with H. pylori 26695; the threshold for the presence of a gene was defined as \u003e0.25. The multidimensional analyses (Genesis software) for the hierarchical clustering as well as for the Principal Component Analysis were performed using the 254 continuous values from the 120 heterologous hybridization experiments, each corresponding to (log10normalized intensity values of strain 26695) minus (log10normalized intensity values of the heterologous strain) (i.e. log26695-logheterol.strain).","divisions":[{"label":"title","span":{"begin":0,"end":44}},{"label":"p","span":{"begin":45,"end":1026}},{"label":"p","span":{"begin":1027,"end":1676}}],"tracks":[{"project":"2_test","denotations":[{"id":"20537153-15504269-10781750","span":{"begin":240,"end":242},"obj":"15504269"},{"id":"20537153-15504269-10781751","span":{"begin":1022,"end":1024},"obj":"15504269"},{"id":"20537153-15504269-10781752","span":{"begin":1899,"end":1901},"obj":"15504269"}],"attributes":[{"subj":"20537153-15504269-10781750","pred":"source","obj":"2_test"},{"subj":"20537153-15504269-10781751","pred":"source","obj":"2_test"},{"subj":"20537153-15504269-10781752","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93d0","default":true}]}]}}