Application of shotgun DNA mapping to yeast genomic DNA

Introduction It is possible to distinguish genomic information based on unzipping DNA with optical tweezers. This is due to the fact that the force signature for unzipping single DNA molecules is sequence-dependent and easily modeled. We can use this information to match an unzipped sequence to a library of sequences obtained through simulations. We call this technique shotgun DNA mapping and have found that we can

use it to better understand protein-DNA interactions and the interaction locations. We currently are pursuing applications in chromatin mapping and structural DNA mapping with many future possible ventures.

Anthony Salvagno, Lawrence Herskowitz, Andy Maloney, Kelly Trujillo, Linh Le, Steve KochUniversity of New Mexico

Acquiring Shotgun Clones

Creating Shotgun ClonesWe started with yeast (S. cerevisiae) and extracted pure genomic DNA. We then digested the genome with both XhoI and EcoRI. After digestion, we ligated the resulting fragments into pBluescript for blue/white screening. We then used E. coli to clone our plasmids with our shot-gun fragments to get shotgun clones. We picked several colonies and combined the remaining colonies to make a “library” of shotgun clones. Some fragments were digested with XhoI only (marked A, C, D, and E)

and others were digested with both XhoI and EcoRI (numbered clones).

What are shotgun clones?Shotgun clones are genomic fragments digested from restriction enzymes and inserted into a cloning vector.

There is no target DNA as every clone used in these experiments is completely random. The randomness of the genomic fragments is key to later elements of shotgun DNA mapping.

Ligating DNA to our unzipping con-struct enables us to unzip target DNA.

pBR322

unzippable

anchor

The image on the left was a �rst attempt at ligating shotgun clones digested with SapI onto the unzipping construct. The image on the right is the most recent attempt at the same ligation.

The clone in the right image is the same as one of the clones in the left image.

Sap14

Sap14

productnot clear 2 distinct

bands

Creating Unzippable DNACreating unzippable DNA requires a 3 piece ligation. The anchor piece is created from PCR of pRL574 and is 1.1kb in length. It is designed with a BstXI site toward the 3’ end. The adapter piece is a short oligo (~20bp) designed with 2 sticky ends: one that is compli-mentary to the anchor and the other which has a SapI/EarI over-hang. Any DNA you want to unzip must have this SapI/EarI over-hang to be the 3rd piece in the ligation.

As a proof of principle, we tested ligation parameters on pBR322. First we digested the plasmid with EarI and gel extracted (digestion results in 2 pieces) the desirable fragment. We then added the frag-ment to our 3 piece ligation and achieved success. We digested our clones with SapI and performed the same ligation on those.

Unzipping DNAWhat do you need to unzip DNA?

In order to unzip there are several components you need: (1) optical tweezers and detection system, (2) unzip-pable DNA and DNA tethers, and (3) software. No component is more important than any other component be-

cause without one we couldn’t perform an experiment.

Our Optical TweezersWe use a 1064nm 4W diode pumped continuous wave laser. We control beam power through the use of an AOM and can manually steer the beam via a one-to-one telescope. We can move the stage through micrometer positioning stages, and for experiments we move the sample with a 1-d piezoelectric stage. We use a quadrant photodiode for beam detection.

dig/anti-dig interaction

biotin/streptavidininteraction

dsDNA anchor

genomic DNA

cover glass surface

Tethering DNAIn order to unzip DNA, we need to be able to pull on it with our tweezers. To do this we must fix DNA to a glass sur-face. This is achieved through digoxygenin-anti-dig inter-actions. The dig molecule is located on the 5’end of the anchor segment of the DNA and we attach anti-dig to glass nonspecifically. We attach 0.51um polystyrene spheres coated with streptavidin to the DNA via a biotinylated nucleotide in the adapter oligo. There is a nick about 8 bases from the biotin and it is this nick that allows us to separate each strand of DNA. The tethering process itself is not trivial and relies on the concentration of the DNA, clean glass, unclumped microspheres, pure anti-dig, and buffer.

no DNA ~400nM dsDNA

~4nM dsDNA~20nM dsDNA

Demonstrating how DNA concentration can a�ect a typical tether-ing experiment. Visually there are more beads in each sample, but the number of apparent stuck beads increase with increasing DNA

concentration.

Open Science

Open Notebook ScienceMy notebook is hosted by OpenWetWare.org and is a wiki environment that allows me to fully customize my notebook. I can embed movies, presentations, spreadsheets, and just about anything. A large portion of my notebook is dedicated to my day-to-day dealings, but I also use it to publish methods, protocols, and data. I publish everything regardless of success or failure. What I publish is instantly accessible to the world and is completely searchable by Google. An added advantage is that I can access my note-book from anywhere in the world, all I need is an internet connection.

Future PlansTelomere Mapping Because the telomere region is made up of highly repetitive DNA, we believe that we can use optical tweezers to detect each repeat and analyze various structures of the telomere region. These experiments could probe G-quadruplexes, Telomerase interactions, scafolding proteins, and more.

RNA Pol II interactions Transcription is a very complicated process especially in higher order organisms. Unzipping through an assembled RNA Pol II complex could reveal a lot of insight into the nature of the enzyme. We believe that we can also unzip through Pol II during various stages of transcription for further insight into the process. It will also be useful to have an unzipping profile when dealing with chromatin mapping in vivo.

some pictures here

Acknowledgements

Mary Ann OsleyPranav RathiBrian Josey

Karen AdelmanCameron Neylon

Jean-Claude BradleyDiego Ramallo

Stefanie Gallegos

References

Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Open-ing DNA with Piconewton Forces. Physical Review Letters , 4489-4492.

Koch, S. J., & et al. (2002). Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix. Biophysical Journal , 1098-1105.

Shundrovsky, A ., & et al. (2006). Probing SWI/SNF Remodeling of the Nucleo-some by Unzipping Single DNA Molecules. Nature Structural and Molecular Biology , 549-554.

Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical Journal , 1335-1346.

This molecule has 17 nearly identical ~200bp repeats

Chromatin Mapping After Shotgun DNA Mapping, we hope to be able to map nucleosome loca-tions by unzipping through histone proteins bound to dsDNA. Locations would be attainable by retaining the initial unzipping forces, allowing the DNA to rezip, and then unzipping the now naked DNA and using these force curves to match to our database of unzipped fragments.

KochLab