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Bioinformatics Probe Design Tutorial: Primer3, BioCarta, ExPASy, KEGG, WIT, MetaCyc

Learn about probe design for gene networks, explore BioCarta, ExPASy, KEGG, WIT, and MetaCyc tools for biochemical pathway visualization and analysis.

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Bioinformatics Probe Design Tutorial: Primer3, BioCarta, ExPASy, KEGG, WIT, MetaCyc

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  1. Introduction to Bioinformatics - Tutorial no. 13 Probe Design Gene Networks

  2. Probe design problem • Given a genome with a set of genes. • Recall that each gene is represented by a probe (a short segment) of the corresponding cDNA. • The length of a probe can be 20, 50, 70! • The probe design problem tries to identify a probe for every gene.

  3. X4 X1 X2 X3 X X X p1 p1 p1 p1 Probe design problem (Informal definition) • Given a genome, for every cDNA (or mRNA) Xi, • a probe pi is identified such that • pi is a substring of Xi; and • pi does not cross-hybridize with other mRNAs • Probe design problem creates a probe pi for every mRNA Xi

  4. Primer3 Input Repeats library Sequence 5’ PCR primer Oligo probe 3’ PCR primer Include in PCR primer Exclude from PCR primer

  5. Primer3 Output Product Length % GC Self complementarity mRNA position Hybrid Tm Sequence 3’ complementarity 5’ PCR primer Oligo probe 3’ PCR primer

  6. OligoDB Sequence name to find

  7. OligoDB Input Oligonucleotide length Number for each mRNA Background database Ensembl Gene ID Ensembl mRNA ID

  8. OligoDB Output mRNA position Tm of hybrid Probe stability Background matches Oligo probe

  9. Biochemical Pathway Visualization Tools • BioCarta • EXPASY • KEGG • WIT • MetaCyc

  10. BioCarta • Main focus: Pathways and tools to study them. • Pathway tool is web-based. http://www.biocarta.com/genes/index.asp • Static pages of pathways. • Metabolic and regulatory pathways. • Need licence to add/edit pathways.

  11. BioCarta Homepage

  12. BioCarta Metabolic Pathway Example • Glycolysis. • Compounds shown in chemical and textual form. • Enzymes above arrows with legend at bottom. • Only enzymes are clickable.

  13. BioCarta Regulatory Pathway Example • ATM signalling pathway. • All genes are clickable.

  14. ExPASy • “Expert Protein Analysis System”. • Main focus: Analysis of protein sequences and structures. • Pathway tool is web-based. http://www.expasy.org/cgi-bin/search-biochem-index • Static pages of pathways. • Metabolic and Regulatory pathways. • Scanned in version of the Boehringer Mannheim “Biochemical Pathways” map. • Map partitioned into 115 pieces. • Keywords matched against entries in map. • Cannot add/edit pathways.

  15. ExPASy Homepage

  16. ExPASy Metabolic Pathway Example • Glycolysis. • Color convention based on Michal’s “Biochemical Pathways”. • Color of compounds depends on compound type. • Color of arrows depend on species (plant, animal, yeast, prokaryotes). • Only enzymes and arrows outside diagram are clickable.

  17. ExPASy Regulatory Pathway Example • Insulin receptors. • Only enzymes and arrows outside diagram are clickable.

  18. KEGG • “Kyoto Encyclopedia of Genes and Genomes”. • Main focus: Biochemical pathway visualization. • Pathway tool is web-based. http://www.kegg.com/kegg/kegg2.html • Static pages of pathways. • Metabolic and Regulatory pathways. • Different organisms.

  19. KEGG Homepage

  20. KEGG Metabolic Pathway Example • Glycolysis. • Substrates and products are drawn in circles. • Enzymes, represented by EC number, are drawn in rectangles. • Adjacent pathways are drawn in semi-rectangles. • Enzymes found in the gene catalog of a specific organism are marked green. • All items are clickable.

  21. KEGG Metabolic Pathway Example Select specific species Link to compound Link to other pathway Link to enzyme

  22. KEGG Regulatory Pathway Example • MAPK signaling pathway (Homo Sapiens).

  23. WIT • “What is There”. • Main focus: Produce metabolic reconstructions for sequenced (or partially sequenced) genomes. • Pathway tool is Web-based. http://wit.mcs.anl.gov/WIT2/ • Static pages of pathways. • Only metabolic pathways.

  24. WIT Homepage

  25. WIT Metabolic Pathway Example • Glycolysis. • Enzymes. Substrates, and products are clickable.

  26. MetaCyc • Electronic encyclopedia of over 450 metabolic pathways from over 150 different organisms. • Software: • Implemented in COMMON LISP; AI techniques incorporated to allow inference, such as pathway prediction from sequenced genomes. • Need a licence to obtain software. • Free for academic purposes only. • Graph layout algorithm for drawing metabolic pathways dynamically at run-time. • Need to manipulate database to add/edit pathways.

  27. MetaCyc Software

  28. MetaCyc Metabolic Pathway Example • Glycolysis. • Everything (Substrates, products, adjacent pathways, and arrows) is clickable.

  29. Question • Do the enzymes form the same pathway share a common regulation? • Inspect Lysine Biosynthesis pathway. • Use the following tools: • KEGG: http://www.genome.jp/kegg/ • UCSC Genome Browser: genome.ucsc.edu • TransFac database or alternative search tools like TESS: http://www.cbil.upenn.edu/tess/

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