Technology for Systems Biology Nucleic Acid Hybridization In

Technology for Systems Biology

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

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

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

® Affymetrix Gene. Chip Probe Arrays Gene. Chip Probe Array Hybridized Probe Cell Single stranded, fluorescently labeled DNA target Oligonucleotide probe * * * 20µm 1. 28 cm Each probe cell or feature contains millions of copies of a specific oligonucleotide probe Over 400, 000 different probes complementary to genetic information of interest Image of Hybridized Probe Array

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

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

Preparatory Steps • • Extraction of nucleic acids Making c. DNA / c. RNA Amplification Shearing

Chromatin Immuno-Precipitation • • Cross-linking Immuno-precipitation Release Hybridization

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

High-thruput Sequencing (Solexa)

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

What you see mi. RNA profiles