technology for systems biology. nucleic acid hybridization in principle complementary strands will...
TRANSCRIPT
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
**
**
*
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
16 18 20 22 24
05000
15000
25000
15:25
16 18 20 22 240
5000
1000015000
15:25
rat.8[ii, 1
:11]
16 18 20 22 24
05000
10000
15000
15:25
rat.8[ii, 1
:11]
16 18 20 22 24
01000
3000
5000
15:25
16 18 20 22 24
01000
2000
3000
4000
15:25
rat.8[ii, 1
:11]
16 18 20 22 24
01000
2000
3000
4000
15:25
rat.8[ii, 1
:11]
0500
1000150020002500
0500
1000
1500
rat.8[ii, 1
:11]
0500
1000
1500
rat.8[ii, 1
:11]
miRNA profiles