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 cDNA clones

• Synthetic oligonucleotides spotted– Ink jet technology

• In-situ synthesis of oligos– Affymetrix: lithographic process– Nimblegen: micromirrors

• Bead Arrays – Illumina

Affymetrix GeneChip® Probe Arrays

Single stranded, fluorescentlySingle stranded, fluorescentlylabeled DNA targetlabeled DNA target

20µm20µm

Each probe cell or feature containsEach probe cell or feature containsmillions of copies of a specificmillions of copies of a specificoligonucleotide probeoligonucleotide probe

Image of Hybridized Probe ArrayImage of Hybridized Probe Array

Over 400,000 different probes Over 400,000 different probes complementary to geneticcomplementary to geneticinformation of interestinformation of interest

Oligonucleotide probeOligonucleotide probe

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1.28cm1.28cm

GeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe Cell

Nimblegen Oligo Arrays

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

Spin-offs of Array TechnologyProtein-binding ds DNA arrays (PBM)

•High-throughput primers

Preparatory Steps

• Extraction of nucleic acids

• Making cDNA / cRNA

• Amplification

• Shearing

Chromatin Immuno-Precipitation

• Cross-linking

• Immuno-precipitation

• Release

• Hybridization

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

High-thruput Sequencing (Solexa)

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

What you see

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miRNA profiles