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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 DNATypical size: 60-mers

Typical length = 60 nts

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Resolution:60 nt probes30 nt overlapping windows

Tiling arrays

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A type IIs restriction enzyme cuts outside its recognition sequences

10BsmFI

GGGACNNNNNNNNNN / NNNNNNNCCCTGNNNNNNNNNNNNNN / NNNN

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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

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

Ab to the protein of interest

Formaldehyde crosslinks can be reversed by heat, pH, or high salt

Using protein A beads

Cy5 and Cy3 are fluorescent labeling compounds of different color

Gives total DNA signal for comparison

No-antibody background

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Formadehyde (HCHO)

Cross-linked chromatin

Isolate nucleiFragment by sonicationAdd antibody, no antibody = control

Immunoprecipitate

Reverse crosslinks (65o)

PCR amplify and label:Cy5 Cy3

Hybridize to microarray

Measure red/green = enrichment by antibody

ChIP-chip for protein binding sites on DNA in vivo

Adapted from http://www.abcam.com/index.html?pageconfig=resource&rid=10738&pid=5

Protein of interest

via linker ligation (ligate a constant DS sequence to all fragments and then do PCR)or random priming(using random hexamers, say)

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1.454 sequencing

Amplify singleDNA molecules on single beads

Sequence eachDNA/bead bystepwise Incorporation ofA, G,C or T in mini-wells

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Aqueous microsphere

bead

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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-attachedTemplates from one original template molecule

Anneal primer for sequencing and loadDNA polymerase and SSB after enrichingFor template-loaded beads

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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

No template

No bead

No template or bead

Had one template

Had another template

Aqueous microspheres

Remove oil

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Big beads- Template, primer, DNA polymerase

Small beads- ATP sulfurylase, Luciferase

Solution- One dNTP Luciferin, APS

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Pyrosequencing

17APS = adenosine phosphosulfate

Destroy old nucleoside triphosphate substrate before adding new one

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20Red, green, blue, pink

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2005

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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)

2. Solexa/Illumina sequencing

Intelligent Bio-Systems (Jue, Turro… Columbia)

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Solexa-Illumina

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3. Applied Biosystems SOLiD sequencing

Shendure, Church et al.

Polony (polymerase colony) by emulsion PCRor similar on beads (BEAMing)Attach beads to glass slide for sequencing

Sequence by ligation!

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.

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ATTACGGC

AACCGGTT

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5 primer roundsIn total

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