1. Technology for Systems Biology

2. Nucleic Acid Hybridization In principle complementary strands will associate Chemistry is quite different on surfaces compared to solution Each sequence has a characteristic ‘melting’ temperature – Tm

3. Variety of Hybridization Assays RNA abundance DNA copy number (CGH) Localization of specific proteins on DNA –Protein-DNA binding –Protein-DNA interaction DNA methylation –Restriction enzyme –Bisulphite conversion RNA binding DNA sequencing

4. Construction of Arrays Spotted cDNA clones Synthetic oligonucleotides spotted –Ink jet technology In-situ synthesis of oligos –Affymetrix: lithographic process –Nimblegen: micromirrors Bead Arrays –Illumina

5. Affymetrix GeneChip ® Probe Arrays Single stranded, fluorescently labeled DNA target 20µm Each probe cell or feature contains millions of copies of a specific oligonucleotide probe Image of Hybridized Probe Array Image of Hybridized Probe Array Over 400,000 different probes complementary to genetic information of interest Oligonucleotide probe * * * * *1.28cm GeneChip Probe Array Hybridized Probe Cell

6. Nimblegen Oligo Arrays Micro-mirrors direct light to mask and unmask free ends

7. Spin-offs of Array Technology Protein-binding ds DNA arrays (PBM) High-throughput primers

8. Preparatory Steps Extraction of nucleic acids Making cDNA / cRNA Amplification Shearing

9. Chromatin Immuno-Precipitation Cross-linking Immuno-precipitation Release Hybridization

10. Cross-hybridization Most specificity / signal at 70 bp cDNA more specific than cRNA Probes with G-G-G stacks show much more cross-hybridization Sources of cross-hybridizing signal: –RNA & DNA Paralogs Nonspecific interactions –DNA Repeats

11. High-thruput Sequencing (Solexa)

12. Advantages and Drawbacks No cross- hybridization Sensitive to even very low copy numbers Insensitive to SNP’s Able to detect unexpected splice variants Cost Labor Possible unknown biases

13. What you see miRNA profiles