Abstract
BackgroundNucleic acid hybridization is an extensively adopted principle in biomedical research, in which the performance of any hybridization-based method depends on the specificity of probes to their targets. To determine the optimal probe(s) for detecting target(s) from a sample cocktail, we developed a novel algorithm, which has been implemented into a web platform for probe designing. This probe design workflow is now upgraded to satisfy experiments that require a probe designing tool to take the increasing volume of sequence datasets.ResultsAlgorithms and probe parameters applied in UPS 2.0 include GC content, the secondary structure, melting temperature (Tm), the stability of the probe-target duplex estimated by the thermodynamic model, sequence complexity, similarity of probes to non-target sequences, and other empirical parameters used in the laboratory. Several probe background options,Unique probe within a group,Unique probe in a specific Unigene set,Unique probe based onthe pangenomic level, and Unique Probe in the user-defined genome/transcriptome, are available to meet the scenarios that the experiments will be conducted. Parameters, such as salt concentration and the lower-bound Tm of probes, are available for users to optimize their probe design query. Output files are available for download on the result page. Probes designed by the UPS algorithm are suitable for generating microarrays, and the performance of UPS-designed probes has been validated by experiments.ConclusionsThe UPS 2.0 evaluates probe-to-target hybridization under a user-defined condition to ensure high-performance hybridization with minimal chance of non-specific binding at the pangenomic and genomic levels. The UPS algorithm mimics the target/non-target mixture in an experiment and is very useful in developing diagnostic kits and microarrays. The UPS 2.0 website has had more than 1,300 visits and 360,000 sequences performed the probe designing task in the last 30 months. It is freely accessible at http://array.iis.sinica.edu.tw/ups/.Screen cast: http://array.iis.sinica.edu.tw/ups/demo/demo.htm
Highlights
Nucleic acid hybridization is an extensively adopted principle in biomedical research, in which the performance of any hybridization-based method depends on the specificity of probes to their targets
Unique probe(s) for detecting target(s) from a sample cocktail, we developed an algorithm and implemented this algorithm in a probe design web platform, the Unique Probe Selector (UPS) [3]
We implemented a user-friendly webbased tool UPS [3] for designing probes with small likelihoods of forming non-specific duplexes to the other submitted targets in the same query batch or to the sequence set of an NCBI/Unigene-listed species, and upgrades its capability for a customized reference sequence set in the current version
Summary
Nucleic acid hybridization is an extensively adopted principle in biomedical research, in which the performance of any hybridization-based method depends on the specificity of probes to their targets. To determine the optimal probe(s) for detecting target(s) from a sample cocktail, we developed a novel algorithm, which has been implemented into a web platform for probe designing This probe design workflow is upgraded to satisfy experiments that require a probe designing tool to take the increasing volume of sequence datasets. The parameters used in UPS include GC content, GC clamps, the duplex stability estimated by thermodynamic theory model, the secondary structure of probes, a low-complexity mask, and other empirical preferences of wet-lab researchers. This probe design tool is able to overcome the problem of background noise during hybridization. We implemented a user-friendly webbased tool UPS [3] for designing probes with small likelihoods of forming non-specific duplexes to the other submitted targets in the same query batch or to the sequence set of an NCBI/Unigene-listed species, and upgrades its capability for a customized reference sequence set in the current version
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