Croning et al. BMC Genomics 2010, 11:74 http://www.biomedcentral.com/1471-2164/11/74 S O F T W A R E Open Access Automated design of genomic Southern blot probes Mike DR Croning, David G Fricker, Noboru H Komiyama, Seth GN Grant* Abstract Background: Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results: We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to experimentally validate a number of these automated designs by Southern blotting. The majority of probes we tested performed well confirming our in silico prediction methodology and the general usefulness of the software for automated genomic Southern probe design. Conclusions: Software and supplementary information are freely available at: http://www.genes2cognition.org/ software/southern_blot Background Southern blotting is a DNA analysis technique that allows one to detect a specific DNA sequence in a com- plex DNA sample [1]. Gel electrophoresis is used to size separate restriction-digested DNA, which is then trans- ferred (or blotted) to a solid support such as a filter for probing and detection by radioactive or luminescent labelling. * Correspondence: sg3@sanger.ac.uk Genes to Cognition Programme, The Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK The method has found widespread application throughout molecular biology. It has been used for gene discovery and mapping. It also has diagnostic and foren- sic applications, such as mutation detection in patient samples and DNA fingerprinting in criminal investiga- tions. It has been employed as the definitive method for detecting transgene integration [2], and successful homologous recombination in gene targeting experi- ments that ablate or modify a gene’s function in vivo [3]. For the technique to succeed one needs to identify a probe sequence that is unique within the genome for the gene or locus of interest so that it does not cross- © 2010 Croning et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.