DNA microarrays Affymetrix chips 25 mers 20 per

DNA microarrays Affymetrix chips: 25 -mers, 20 per m. RNA sequence (to average out different hybridization efficiencies) Oligonucleotides synthesized in place using photolithography (light +/- masks) Grown sequences 1

Nimblegen: addressable micro-mirrors to deprotect small spots of growing DNA Typical size: 60 -mers Typical length = 60 nts 2

Resolution: 60 nt probes 30 nt overlapping windows Tiling arrays 3

A type IIs restriction enzyme cuts outside its recognition sequences Bsm. FI 10 GGGACNNNNN / NNNNNNN CCCTGNNNNNNN / NNNN 14 4

SAGE (serial analysisof gene expression) =Nla. III 10 bases downstream on the top strand 5

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Ch. IP-chip: for protein – DNA interactions Isolate chromatin Formaldehyde (HCHO) crosslinks amino groups on proteins to functional groups on DNA bases No-antibody background Ab to the protein of interest Using protein A beads Formaldehyde crosslinks can be reversed by heat, p. H, or high salt Gives total DNA signal for comparison Cy 5 and Cy 3 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) 7

Ch. IP-chip for protein binding sites on DNA in vivo Protein of interest Formadehyde (HCHO) Cross-linked chromatin Isolate nuclei Fragment by sonication Add antibody, no antibody = control Immunoprecipitate Reverse crosslinks (65 o) via linker ligation (ligate a constant DS sequence to all fragments and then do PCR) or random priming (using random hexamers, say) PCR amplify and label: Cy 5 Cy 3 Hybridize to microarray Adapted from http: //www. abcam. com/index. html? pageconfig=resource&rid=10738&pid=5 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 9

bead Aqueous microsphere 10

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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. 13

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 bead Remove oil 14

Big beads- Template, primer, DNA polymerase Small beads- ATP sulfurylase, Luciferase Solution- One d. NTP Luciferin, APS 15

Pyrosequencing 16

Destroy old nucleoside triphosphate substrate before adding new one APS = adenosine phosulfate 17

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Red, green, blue, pink 20

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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 d. NTPs (one nucleotide at a time) 22

Solexa-Illumina 23

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3. Applied Biosystems SOLi. D 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. , Mc. Cutcheon, 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! 31

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AT TA CG GC AA CC GG TT 37

5 primer rounds In total 38

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