DNA microarrays
Affymetrix chips:
25-mers, 20 per mRNA sequence (to average out different hybridization efficiencies)
Oligonucleotides synthesized in place using photolithography (light +/- masks)
Grown sequences
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Nimblegen : addressable micro-mirrors to deprotect small spots of growing DNA
Typical size: 60-mers
Typical length = 60 nts
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Resolution:
60 nt probes
30 nt overlapping windows
Tiling arrays
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SAGE (serial analysisof gene expression)
=NlaIII
10 bases downstream on the top strand
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ChIP-chip: for protein – DNA interactions
Ab to the protein of interest
Using protein A beads
Formaldehyde crosslinks can be reversed by heat, pH, or high salt
Cy5 and Cy3 are fluorescent labeling compounds of different color via linker ligation
(ligate a constant DS sequence to all fragments and then do PCR) or random priming
(using random hexamers, say)
Isolate chromatin
Formaldehyde (HCHO) crosslinks amino groups on proteins to functional groups on
DNA bases
No-antibody background
Gives total DNA signal for comparison
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ChIP-chip for protein binding sites on DNA in vivo via linker ligation
(ligate a constant DS sequence to all fragments and then do PCR) or random priming
(using random hexamers, say)
Protein of interest
Formadehyde (HCHO)
Cross-linked chromatin
Isolate nuclei
Fragment by sonication
Add antibody, no antibody = control
Immunoprecipitate
Reverse crosslinks (65 o )
Cy5
PCR amplify and label:
Cy3
Adapted from http://www.
abcam.com/index.html?pageconfig=resource&rid=10738&pid=5
Hybridize to microarray
Measure red/green = enrichment by antibody 8

1.
454 sequencing
Amplify single
DNA molecules on single beads
Sequence each
DNA/bead by stepwise
Incorporation of
A, G,C or T in mini-wells
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bead
Aqueous microsphere
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BEAMing: PCR on beads compartmentalized in a water-oil emulsion.
Millions of primers attached to each bead,
Producing millions of copies of bead-attached
Templates from one original template molecule
Anneal primer for sequencing and load
DNA polymerase and SSB after enriching
For template-loaded beads 12

Attached oligomers were pre-labeld red or green, then mixed and emulsified.
See single beads in aqueous microspheres in oil.
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BEAMing = beads, amplification, emulsion, magnetics
= cloning DNA molecules via PCR on beads
Aqueous microspheres
No template or bead
Had one template
Had another template
No template
No bead
Remove oil
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Big beadsTemplate, primer,
DNA polymerase
Small beads- ATP sulfurylase,
Luciferase
SolutionOne dNTP
Luciferin, APS
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Pyrosequencing
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Destroy old nucleoside triphosphate substrate before adding new one
APS = adenosine phosphosulfate
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blue
pink
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2. Solexa/Illumina sequencing
Intelligent Bio-Systems (Jue, Turro… Columbia)
Amplification in situ on glass surface of flow cell
(PCR that keeps different DNAs separate- “micro-cloning”
Sequencing with reversible fluorescent terminator dNTPs
(one nucleotide at a time)
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3. Applied Biosystems SOLiD sequencing
Shendure, Church et al.
Webinar: http://appliedbiosystems.cnpg.com/lsca/webinar/rhodes/chemistry/20070618/
Shendure, J., Porreca, G.J., Reppas, N.B., Lin, X., McCutcheon, J.P., Rosenbaum, A.M., Wang,
M.D., Zhang, K., Mitra, R.D., and Church, G.M. 2005. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309: 1728-1732.
Polony (polymerase colony) by emulsion PCR or similar on beads (BEAMing)
Attach beads to glass slide for sequencing
Sequence by ligation!
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AA
CC
GG
TT
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