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Integrated Design Flow for Universal DNA Tag Arrays N. 1 Hundewale , 1CS – In which cell tissues and under what environmental conditions is each gene active? – How does the activity level of a gene change with: cell cycle stage, environmental conditions, disease, etc.? – What genes seem to be regulated together? • Universal tag arrays (UTAs) technology – Provides unprecedented assay customization flexibility while maintaining a high degree of multiplexing and low unit cost • In this poster we describe an integrated design flow for genomic assays based on UTAs – We use the proposed flow to design UTA-based assays for measuring Herpes B viral gene expression in cells derived from macaque and human hosts – After defining a “B virus molecular signature”, the assay can provide a sensitive tool for early B virus infection diagnosis and differentiation between B herpes and the closely related herpes simplex viruses Design Flow • “Programmable” Array Format [Brenner 97, Morris et al. 98] – Array consists of application independent oligonucleotides called tags – Two-part reporter probes: aplication specific primers ligated to antitags – Detection carried by a sequence of reactions separately involving the primer and the antitag part of reporter probes • Tag/Antitag Hybridization Constraints (H1) Antitags hybridize strongly to complementary tags (H2) No antitag hybridezes to a non-complementary tag (H3) Antitags do not cross-hybridize to each other t1 t1 t2 t2 t1 t1 t2 • Open reading frames (ORFs) Bioperl GenMark/ ORF Finder ORFs in Fasta format Promide PerTags and A. 1 Zelikovsky ORF and Primer Selection Genomic IDs Probe pools Tag/antitag sequences C. 2 Prajescu , Universal DNA Tag Arrays • DNA microarrays provide a tool for answering a wide variety of questions about the dynamics of cells Assay parameters L. 3 Perelygina , Department, GSU, 2CSE Department, UCONN, 3Department of Biology, GSU Abstract Sequences in FASTA format I. 2 Mandoiu , PrimerDel+ Reporter probes – ORFs are regions of genetic material beginning with a start codon and ending with a stop codon that might code for a protein – ORFs can be extracted by means of the genome's sequence or id using ORF Finder. A second approach is to use the GenMark family of statistical gene prediction programs [Borodovsky] •Primer selection -Constraints: - Homogeneity: Each primer must hybridize to its target site at the temperature selected for the experiment - Sensitivity: Must avoid self-hybridization and ensure that primers do not form secondary structures - Specificity: Each primer must hybridize to one particular ORF -Selection tools: Hybridization Experiment and Analysis - Primer and microarray probe selection are well studied; we use the Promide tool [Rahmann 03] for selecting pools of primer candidates meeting the above constraints for each ORF Conclusions • We have described a suite of software tools for designing genomic assays based on UTAs – Integrating design flow optimization steps yields higher multiplexing rates and leads to reduced assay costs • In future work we will make the entire software suite available as an online web server • • • • • • • • • References Aymetrix, Inc., GeneFlex tag array probe set, available at the NetAffx™ Analysis Center, http://www.affymetrix.com/analysis/ M. Atlas, N. Hundewale, L. Perelygina, and A. Zelikovsky, Proc. International Conf. of the IEEE Engineering in Medicine and Biology (EMBC), pp. 172-175, 2004. A. BenDor, T. Hartman, B. Schwikowski, R. Sharan, and Z. Yakhini. Towards optimally multiplexed applications of universal DNA tag systems. Proc. 7th Annual International Conference on Research in Computational Molecular Biology (RECOMB), pp. 48-56, 2003 S. Brenner. Methods for sorting polynucleotides using oligonucleotide tags. US Patent 5,604,097, 1997. I.I. Mandoiu and D. Trinca. Exact and approximation algorithms for DNA tag set design. Proc. 16th Annual Symposium on Combinatorial Pattern Matching (CPM), pp. 383-393, 2005. I.I. Mandoiu, C. Prajescu, and D. Trinca. Improved tag set design and multiplexing algorithms for universal arrays. Proc. 5th Int. Conf. on Computational Science (ICCS 2005), Part II, pp. 994-1002, 2005. M. Borodovsky, Genemark, http://opal.biology.gatech.edu/GeneMark ORF finder, http://www.ncbi.nih.gov/gorf/gorf.html. S. Rahmann, Rapid large-scale oligonucleotide selection for microarrays, Proc. IEEE Computer Society Bioinformatics Conference (CSB), 2002. RECOMB 2005, Poster Session A, Bay 43