Probe design is the most important step for any microarray based assay. Accurate and efficient probe design and selection for the target sequence is critical in generating reliable and useful results. Several different approaches for probe design are reported in literature and an increasing number of bioinformatics tools are available for the same. However, based on the reported low accuracy, determining the hybridization efficiency of the probes is still a big computational challenge. Present study deals with the extraction of various novel features related to sequence composition, thermodynamics and secondary structure that may be essential for designing good probes. A feature selection method has been used to assess the relative importance of all these features. In this paper, we validate the importance of various features currently used for designing an oligonucleotide probe. Finally, a classification methodology is presented that can be used to predict the hybridization quality of a probe.

UNLABELLED: PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.edu