Technology for Systems Biology

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Technology for Systems Biology
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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

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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

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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

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Affymetrix GeneChip Probe Arrays
Single stranded, fluorescently labeled DNA target
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Nimblegen Oligo Arrays

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

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

• High-throughput primers

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Preparatory Steps

• Extraction of nucleic acids
• Making cDNA / cRNA
• Amplification
• Shearing

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Chromatin Immuno-Precipitation

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

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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

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High-thruput Sequencing (Solexa)
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Advantages and Drawbacks

• No cross-hybridization
• Sensitive to even very low copy numbers
• Insensitive to SNPs
• Able to detect unexpected splice variants

• Cost
• Labor
• Possible unknown biases

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What you see
miRNA profiles