Croning et al. BMC Genomics 2010, 11:74 http://www.biomedcentral.com/1471-2164/11/74 Page 5 of 7 Figure 3 Relationship between genomic design window size and the number of candidate probes generated by tiling. The relationship is found to be linear. Using a 3 kb input window and acceptable probe length in the range 500-1300 bp, approximately 900 candidate probes are generated. Points lying off the main line arise from probe design searches where the tiling granularity has been reduced to try to find a suitable probe in difficult to design loci. Each of these programs read their customizable para- meters from a .ini type configuration file. Results and Discussion To date we have designed 124 probes using flanking regions in about 60 genes that we chose to perturb by gene targeting in mouse embryonic stem cells. Given a ~3 kb window in which to search for a Southern blot probe and a desirable length range for the final probe of 500- 1300 bp, the tiling strategy outlined produces on average ~900 candidate probes (when used with the default granu- larity) to search against the genome (see Figures 1 and 3). In total 103/124 (83%) of these designs passed by the criteria above of score ratio ≥ 10 and repetitive/low- complexity DNA content ≤ 5%. On average the best candidate probes for each design were 818.1 ± 25.0 bp long and contained only 4.1 ± 1.0% repetitive and low complexity DNA, the latter being significantly lower than our manually-designed calibration set (p < 0.05, Student’s t-test, Table 2). Additionally by such brute-force searching and scoring of genomic probes, in exactly half the design cases (62) it was possible to find one or more unique probes amongst those tested. These had a single (i.e. ideal) hit to their target genomic locus with no cross-reactivity to other loci. It is worth noting that none of the calibration probes gave this ‘ideal’ result, when evaluated using the same exonerate search parameters. The remaining probes (97/124) that passed our empirical cut-off criteria, had an average score-ratio of 23.7 ± 1.3, which was not significantly different to the experimental calibration set (p > 0.05; Student’s t-test). In order to confirm the system does design effective Southern blot probes we experimentally tested 16 of the in silico designs. Blots were performed on mouse geno- mic DNA extracted from embryonic stem cell lines in Table 2 Comparison of manually-designed (calibration) and automatically-designed Southern blot probes. Probe set Length (bases) Calibration 791.6 ± 85.9 (n = 8) Automated 818.1 ± 25.0 (n = 124) Average score ratio (self/ second hit) 19.5 ± 10.8 23.7 ± 1.3 Average repetitive & low- complexity DNA (%) 18.2 ± 10.8 4.1 ± 1.0* Unique hit to genome 0/8 62/86 Passed empirical selection criteria 3/8 103/134 Shown are the average length (± standard error) of the probes in the calibration and automatically designed sets, together with their average score ratio, which is calculated as the ratio of the score of the on-target hit of the probe to the genomic locus of interest, and the highest-scoring off-target second best hit. The average combined percentage repetitive and low-complexity DNA content (as estimated by RepeatMasker and DUST) is shown for each of the probes. Also shown are the number of probes in each set that had a unique hit to their target genomic locus, with no off-target hits i.e. no apparent cross-reactivity. * p < 0.05 Student’s t-test.