0.5 – 1 μg of high-purity genomic DNA, which may be difficult to obtain from all samples. To overcome this limitation the potential for DNA amplification, artefacts that may significantly alter hybridization to the microarray were examined. To analyze for this possible limitation, AZD8186 order a 10 ng (4.89 × 106 copies) aliquot of Francisella tularensis LVS strain genomic DNA [Accession number NC_007880, genome size 1,895,994 bases] was amplified using the whole genome amplification method (GenomiPhi V2, GE Healthcare). A total of 1 μg of the resulting amplified DNA was hybridized to the UBDA array and compared to the hybridization pattern resulting from the hybridization of 1 μg of unamplified DNA from the same source. Figure 6 shows a linear regression of the two samples (all 262,144 probes) which resulted in an R2 value of 0.91, well within the R2 = 0.94 +- 0.06 reproducibility click here found for the custom microsatellite microarray . This confirms that whole genome amplification of pathogen material in small amounts
is comparable to the unamplified genomic sample. We obtained these results using the standard protocol with 10 ng of starting material without optimization. We are targeting a 1-2 nanogram sample size as a starting amount of material in an optimized robust, field sample evaluation. Figure 6 Bivariate Fit of Francisella tularensis whole genome amplified genomic DNA (log 2 values) by unamplified genomic DNA (log 2 values). A linear regression of the two samples resulted in an R2 value
of 0.91, confirming that whole genome amplification of pathogen material such as Francisella tularensis LVS genomic DNA in small amounts (10 ng starting material) is comparable to the unamplified genomic sample. Discussion This is a new forensics array based technology to identify any species. This unique strategy of using patterns generated from hybridization of any unknown genome (DNA or cDNA) to a very mafosfamide selleck inhibitor high-density species independent oligonucleotide microarray and comparing those patterns to a library of patterns of known samples can be used to identify unknown organisms. Figure 5 shows the grouping of the different genomes into bacterial, viral and eukaryotic genomes. Further the Brucella species grouping pattern obtained from the phylogenomic analysis using the Pearson’s correlation matrix shown in Figure 5 are in agreement with Brucella species showing hierarchical clustering represented as a similarity matrix shown in Figure 3. The UBDA hybridization patterns are unique to a genome, and potentially to different isolates and to a mixture of organisms. In the future, this forensics method will work by comparing signal intensity readout to a library of readouts established by interrogating a wide spectrum of species which will be available at our website http://discovery.vbi.vt.edu/ubda/. The phylogenetic tree illustrates the ability of 9-mer probes to differentiate among Brucella species.