1 / 53

Pre-genomic era: finding your own clones

Pre-genomic era: finding your own clones. Imagine initial cDNA or PCR fragment probe hybridizes to clones 3, 9, 16, 22. GENOME. 1. 2. 3. 23. CHROMOSOMES. YACs. or. BACs (map). Plasmid Sub-clones. Gridded (arrayed/ordered) library. 18,000 BACs for Drosophila. 300,000 BACs for Humans.

sakura
Download Presentation

Pre-genomic era: finding your own clones

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Pre-genomic era: finding your own clones

  2. Imagine initial cDNA or PCR fragment probe hybridizes to clones 3, 9, 16, 22

  3. GENOME 1 2 3 23 CHROMOSOMES YACs or BACs (map) Plasmid Sub-clones

  4. Gridded (arrayed/ordered) library

  5. 18,000 BACs for Drosophila 300,000 BACs for Humans

  6. STS- Sequence Tagged Site Mapping Probe or PCR product STS

  7. Minimal Tiling Path

  8. Sub-clone into smaller segments and map Or use primer walking- NOT EFFICIENT Instead use shotgun sequencing Plasmids Sequence >700nt from each end

  9. Virtual DNA (sequence) Assembly

  10. Shotgun Sequencing 2 or 3 libraries of Different size fragments

  11. Mates: read-pairs 2kb library of clones 10kb library of clones

  12. * * * * * * * *

  13. GENOME 1 2 3 23 CHROMOSOMES YACs or BACs (map) Plasmid Sub-clones Don’t map Sequence ends Assemble sequence of BAC

  14. Mates: read-pairs 2kb library of clones 10kb library of clones 150kb library of clones

  15. Cosmids, YACs ordered C. Elegans 100Mb Drosophila 120Mb BACs ordered- STS mapping + fingerprinting Whole Genome shotgun Human 3, 200Mb BACs ordered- fingerprinting Whole Genome shotgun

  16. 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

  17. bead Aqueous microsphere

  18. 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

  19. Attached oligomers were pre-labeld red or green, then mixed and emulsified. See single beads in aqueous microspheres in oil.

  20. BEAMing = beads, amplification, emulsion, magnetics = cloning DNA molecules via PCR on beads No template or bead Aqueous microspheres Had one template Had another template No template No bead Remove oil

  21. Big beads- Template, primer, DNA polymerase Small beads- ATP sulfurylase, Luciferase Solution- One dNTP Luciferin, APS

  22. Pyrosequencing

  23. Destroy old nucleoside triphosphate substrate before adding new one APS = adenosine phosphosulfate

  24. Red, green, blue, pink

  25. 2005

  26. 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)

  27. Solexa-Illumina

  28. 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!

  29. AA CC GG TT AT TA CG GC

More Related