1. My 10-minute presentation… I’m super-serious! (Hopefully) By And KochLab This presentation will include amazing topics like: The Latest in Tweezers Technology Biological Advancements And more…

2. Thank You! KochLab Guys Steve Koch – Lab Don Larry Herskowitz – programmer guy Andy Maloney – optics Guy Linh Le –undergrad guy Nas Manole – undergrad guy Brandon Beck – former biology guy Osley Lab People Mary Ann Osley – head honcho Kelly Trujillo – mentor guy Another Awesome Person Susan Atlas – got us a kick ass grant

3. Last Time… The American Science Classic Revised and Expanded With Over 4,500 Experiments And 1,000 Informative Illustrations THE ALL-PURPOSE KOCHBOOK OF KOCHING by ANTHONY SALVAGNO and KOCHLAB Recap: Prototype optical tweezers Chromatin biology Prelude to Shotgun DNA Mapping

4. Before After Green (532nm) Laser Unstable Beam Successful Trapping Red (690nm) laser Built from components More adaptable system Unsuccessful trapping  Optical Tweezer Progress

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. Biological Progress (Before) (not same picture as earlier) 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

7. Biological Progress (After) New Aims: Shotgun DNA Mapping Unzipping of RNA during elongation Shotgun Chromatin Mapping Telomere Analysis ALL UTILIZING OT!

8. Shotgun DNA Mapping F F

9. Extraction of DNA plasmid linearized plasmid Restriction enzyme Restriction enzyme chromosome Genomic DNA Fragments Combine for tons 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

10. Unzipping of RNA Pol II RNA Pol II Transcription Reassembled Nucleosomes promoter cryptic promoter RNA Pol II is antisymetric can tell difference between sense and antisense. Once force signature is established, we can input into library for SCM. Other Studies: Promoter-proximal stalling Initiation complex analysis Understanding of stalling and termination behavior More questions out there

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. 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. Thanks for listening Kochlab.blogspot.com Openwetware.org/wiki/User:Anthony_Salvagno Openwetware.org/wiki/Koch_Lab