pre-genomic era: finding your own clones
Post on 19-Jan-2016
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Pre-genomic era: finding your own clones
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Imagine initial cDNA or PCR fragment probe hybridizes to clones 3, 9, 16, 22
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GENOME
CHROMOSOMES
YACsor
BACs (map)
PlasmidSub-clones
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Gridded (arrayed/ordered) library
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18,000 BACs for Drosophila
300,000 BACs for Humans
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STS- Sequence Tagged Site Mapping
STS Probe orPCR product
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Minimal Tiling Path
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Sequence >700ntfrom each end
Plasmids
Sub-clone into smaller segments and mapOr use primer walking- NOT EFFICIENT
Instead use shotgun sequencing
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Virtual DNA(sequence)Assembly
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2 or 3 libraries ofDifferent size fragments
ShotgunSequencing
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Mates: read-pairs
2kb library of clones
10kb library of clones
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**
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* * **
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123 23
GENOME
CHROMOSOMES
YACsor
BACs (map)
PlasmidSub-clonesDon’t map Sequence ends
Assemble sequence of BAC
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Mates: read-pairs
2kb library of clones
10kb library of clones
150kb library of clones
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C. Elegans 100Mb
Drosophila 120Mb
Human 3, 200Mb
Cosmids, YACs ordered
BACs ordered- STS mapping+ fingerprinting
BACs ordered- fingerprinting
Whole Genome shotgun
Whole Genome shotgun
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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
30APS = adenosine phosphosulfate
Destroy old nucleoside triphosphate substrate before adding new one
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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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