My 10-minute presentation… I’m
      super-serious!                  (Hopefully)




This presentation will include
amazin...
Thank You!
KochLab Guys                       Osley Lab People
• Steve Koch – Lab Don             • Mary Ann Osley – head ...
Last Time…
                               Recap:
                               •Prototype optical
THE ALL-PURPOSE KOCHBOO...
Optical Tweezer Progress
        Before                   After
                          •Red (690nm) laser
•Green (532nm...
Biological Background
       •DNA is important to a lot of processes in the
       cell
       •Histones compact DNA for s...
Biological Progress (Before)

             •Focus on histone remodeling using optical
             tweezers
             •...
Biological Progress (After)

New Aims:
•Shotgun DNA Mapping
•Unzipping of RNA during
elongation
•Shotgun Chromatin Mapping...
Shotgun DNA Mapping


         F


  F
Extraction of DNA
                                 Steps:
                                 1. Destroy yeast and take DNA
 ...
Unzipping of RNA Pol II
RNA Pol II is antisymetric
    •can tell difference between sense and
    antisense.
Once force si...
Shotgun Chromatin Mapping
In principle, very similar to SDM:
1. Unzip non-naked DNA
2. Pop off nucleosomes and
    polymer...
Future Experiments
• Utilize SDM/SCM for cool stuff
  – Detection of inversions, deletions, …
  – Alternative Splicing
• T...
Thanks for listening

         Kochlab.blogspot.com

Openwetware.org/wiki/User:Anthony_Salvagno
     Openwetware.org/wiki/...
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NSMS IGERT Nano Cafe 2/12/09

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This is a talk I gave at the Nano Cafe in February. It is a quick overview of the things I am doing in KochLab and some of the things we hope to achieve.

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NSMS IGERT Nano Cafe 2/12/09

  1. 1. My 10-minute presentation… I’m super-serious! (Hopefully) This presentation will include amazing topics like: •The Latest in Tweezers Technology By •Biological Advancements •And more… And KochLab
  2. 2. Thank You! KochLab Guys Osley Lab People • Steve Koch – Lab Don • Mary Ann Osley – head honcho • Larry Herskowitz – programmer • Kelly Trujillo – mentor guy guy • Andy Maloney – optics Guy Another Awesome Person • Linh Le –undergrad guy • Susan Atlas – cool kinesin project • Nas Manole – undergrad guy • Brandon Beck – former biology guy • Diego Ramallo – KochLab alumni
  3. 3. Last Time… Recap: •Prototype optical THE ALL-PURPOSE KOCHBOOK tweezers •Chromatin biology •Prelude to Shotgun DNA Mapping OF KOCHING • The American Science Classic • Revised and Expanded • With Over 4,500 Experiments • And 1,000 Informative Illustrations by ANTHONY SALVAGNO and KOCHLAB
  4. 4. Optical Tweezer Progress Before After •Red (690nm) laser •Green (532nm) Laser • Built from components •Unstable Beam •More adaptable system •Successful Trapping  •Unsuccessful trapping 
  5. 5. Biological Background •DNA is important to a lot of processes in the cell •Histones compact DNA for storage and regulation •Histone remodification needed for control of cellular processes like: •Protein synthesis •replication
  6. 6. Biological Progress (Before) •Focus on histone remodeling using optical tweezers •Shundrovsky et al. proved nucleosome location can be found (resolution of 3bp) •We want to map nucleosome locations throughout a gene •Use PHO5 gene because well documented locations (proof of principle) •Getting Chromatin from yeast cells prove to be very difficult (not same picture as earlier)
  7. 7. Biological Progress (After) New Aims: •Shotgun DNA Mapping •Unzipping of RNA during elongation •Shotgun Chromatin Mapping •Telomere Analysis ALL UTILIZING OT!
  8. 8. Shotgun DNA Mapping F F
  9. 9. Extraction of DNA Steps: 1. Destroy yeast and take DNA 2. Use RE to cut DNA into fragments 3. Use plasmids to create clones 4. Attach DNA to tethering construct 5. Unzip and match to library plasmid linearized plasmid Restriction enzyme Combine for tons of DNA Restriction enzyme chromosome Genomic DNA Fragments
  10. 10. Unzipping of RNA Pol II RNA Pol II is antisymetric •can tell difference between sense and antisense. Once force signature is established, we can input into library for SCM. Reassembled Nucleosomes RNA Pol II promoter cryptic promoter Transcription Other Studies: •Promoter-proximal stalling •Initiation complex analysis •Understanding of stalling and termination behavior •More questions out there
  11. 11. Shotgun Chromatin Mapping In principle, very similar to SDM: 1. Unzip non-naked DNA 2. Pop off nucleosomes and polymerases 3. Allow DNA to reaneal 4. Unzip naked DNA 5. Determine locations of popped proteins on DNA fragment 6. Determine location of fragment within genome using SDM library (Larry) 7. Perform several times to get precise locations of nucleosomes and polymerases High throughput for human genome adaptation
  12. 12. Future Experiments • Utilize SDM/SCM for cool stuff – Detection of inversions, deletions, … – Alternative Splicing • Telomere Mapping – Cancer research related – Aging related – Loaded with repeat sequences – Lots can be done with OT
  13. 13. Thanks for listening Kochlab.blogspot.com Openwetware.org/wiki/User:Anthony_Salvagno Openwetware.org/wiki/Koch_Lab

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