topic 9.ppd
TRANSCRIPT
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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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