Croning et al. BMC Genomics 2010, 11:74 http://www.biomedcentral.com/1471-2164/11/74 hybridize with other endogenous DNA sequences pre- sent in the sample. Like others we have routinely used a manual approach to design and test our probes, which is labor intensive and usually requires trial of different probes before the desired result is obtained. It is there- fore highly desirable to have bioinformatic tools that aid in the design process and can also optimize the probes. The typical manual approach is to choose a probe of at least 300 bp in length, to ensure efficient labeling in the random priming reaction [4], and in practice probes of 500-1000 bp are generally employed. Following iden- tification of the genomic locus one wishes to probe, the DNA sequence from a genome browser (such as Ensembl [5]) is examined for repetitive sequence ele- ments as these can result in an intense background smear on hybridization that obscures single copy gene hybridization signals. The test probe is then searched against the genome using BLAST [6] or other means and the results inspected. One hopes to obtain a single perfect match to the target locus on the genome, with little or no cross-reactivity to other loci. If this is not the case, one has to return to the genome browser and move and/or shorten the sequence before repeating the BLAST search. With each genome search taking several minutes this is a time consuming exercise and is unli- kely to yield the best possible probe. Clearly this method is amenable to bioinformatic automation. Many programs already exist for oligonu- cleotide probe discovery, principally in the area of microarray design [7]. These programs are generally designed to find probes less than 100 bp rendering them inapplicable for the considerably longer Southern blot probes. To address this need we have written a sys- tem to find (near) unique probes in a specified region of a genome, which contain little or no repetitive DNA sequence, and also to design PCR primers to facilitate the recovery of the probes from cellular DNA for subse- quent Southern blotting. We went on to experimentally validate a number of these designs by Southern blotting in the mouse genome. Implementation Given user-supplied chromosomal coordinates, and a desirable size range for the southern blot probe (default 500-1300 bp), we used a tiling approach to generate many possible probes in the specified design window. The program starts from the maximum allowable probe length, tiling the window by moving by a small percen- tage of the probe length each time (default 5%). Once this is completed the probe length is reduced by 50 bases (configurable) and the window re-tiled, generating more candidate probes. The process is repeated until the minimum probe length is reached (see Figure 1). The candidate probes are searched against the target Page 2 of 7 genome using the Exonerate pairwise sequence align- ment program [8]. We calibrated the method using a set of manually- designed genomic probes that we have previously suc- cessfully employed for Southern blotting (see Table 1). These had an average length of ~800 bp and when searched with Exonerate (with parameters –model affine:local –score 150) all of these produced a perfect match to their genomic locus (as would be expected) and a number of additional lower-scoring alignments to other loci. On average these second best matches spanned 17.5 ± 5.8% (mean ± standard error) of the probe length, with 74.6 ± 3.2% DNA sequence identity (n = 8). From the scores of the on-target or ‘self-hit’ and the highest scoring off-target locus alignments we calcu- lated a score ratio as measure of uniqueness of the can- didate probe. Our calibration probes averaged 19.5 ± 3.6 (n = 8). This score ratio is proportional to both the length and sequence identity of the two matches. Comparing the probe sequences to a version of the genomic assembly that has been screened for repeats and low-complexity regions by RepeatMasker [9] and DUST [10] allows us to estimate the repetitive DNA content of individual probes. Our calibration probes contained 18.2 ± 10.8% such DNA. Considering these results we chose a minimum score ratio of 10 and a maximum combined repetitive and low-complexity base content of 5% as the minimum requirements for probe acceptance (configurable). Figure 1 Tiling approach to Southern blot probe design. Many potential probes are generated and tested in the user-specified genomic window, starting from the maximum allowable probe length (default 1300), tiling the window by moving by a small percentage of the probe length each time (default 5%). Once this is completed the probe length is reduced by 50 bases (configurable) and the window re-tiled generating more candidate probes. The process is repeated until the minimum probe length is reached (default 500).