1.
Application of shotgun DNA mapping to yeast genomic DNA
Anthony Salvagno, Lawrence Herskowitz, Andy Maloney, Kelly Trujillo, Linh Le, Steve Koch
University of New Mexico
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.
Acquiring Shotgun Clones Unzipping DNA Open Science
What are shotgun clones? What do you need to unzip DNA?
Shotgun clones are genomic fragments digested from restriction enzymes and inserted into a cloning vector. In order to unzip there are several components you need: (1) optical tweezers and detection system, (2) unzip-
There is no target DNA as every clone used in these experiments is completely random. The randomness of the pable DNA and DNA tethers, and (3) software. No component is more important than any other component be-
genomic fragments is key to later elements of shotgun DNA mapping. cause without one we couldn’t perform an experiment.
Creating Shotgun Clones
We 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 Our Optical Tweezers
resulting fragments into pBluescript for We use a 1064nm 4W diode pumped continuous wave laser.
blue/white screening. We then used E. We control beam power through the use of an AOM and can
coli to clone our plasmids with our shot- manually steer the beam via a one-to-one telescope. We can
gun fragments to get shotgun clones. We move the stage through micrometer positioning stages, and for
picked several colonies and combined the experiments we move the sample with a 1-d piezoelectric
remaining colonies to make a “library” of stage. We use a quadrant photodiode for beam detection.
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).
Creating Unzippable DNA
Creating 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
Tethering DNA Open Notebook Science
unzippable In order to unzip DNA, we need to be able to pull on it with
short oligo (~20bp) designed with 2 sticky ends: one that is compli-
our tweezers. To do this we must fix DNA to a glass sur-
My notebook is hosted by OpenWetWare.org and is a wiki environment that allows me
pBR322 mentary to the anchor and the other which has a SapI/EarI over- to fully customize my notebook. I can embed movies, presentations, spreadsheets, and
face. This is achieved through digoxygenin-anti-dig inter-
hang. Any DNA you want to unzip must have this SapI/EarI over-
hang to be the 3rd piece in the ligation.
actions. The dig molecule is located on the 5’end of the no DNA ~400nM dsDNA
just about anything. A large portion of my notebook is dedicated to my day-to-day
anchor segment of the DNA and we attach anti-dig to glass
nonspecifically. We attach 0.51um polystyrene spheres
dealings, but I also use it to publish methods, protocols, and data. I publish everything
As a proof of principle, we tested ligation parameters on pBR322. regardless of success or failure. What I publish is instantly accessible to the world and is
coated with streptavidin to the DNA via a biotinylated
First we digested the plasmid with EarI and gel extracted (digestion
results in 2 pieces) the desirable fragment. We then added the frag-
nucleotide in the adapter oligo. There is a nick about 8 completely searchable by Google. An added advantage is that I can access my note-
bases from the biotin and it is this nick that allows us to
anchor ment to our 3 piece ligation and achieved success. We digested our
separate each strand of DNA. The tethering process itself is
book from anywhere in the world, all I need is an internet connection.
clones with SapI and performed the same ligation on those.
not trivial and relies on the concentration of the DNA, clean ~20nM dsDNA ~4nM dsDNA
glass, unclumped microspheres, pure anti-dig, and buffer.
Ligating DNA to our unzipping con-
Demonstrating how DNA concentration can a ect a typical tether-
struct enables us to unzip target DNA. ing experiment. Visually there are more beads in each sample, but
the number of apparent stuck beads increase with increasing DNA
concentration.
Sap14
Sap14
genomic DNA
product
not clear
2 distinct
biotin/streptavidin
bands interaction
dsDNA anchor
dig/anti-dig
The image on the left was a rst attempt at ligating shotgun clones digested with SapI onto the interaction
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. cover glass surface
Future Plans Acknowledgements References
Telomere Mapping Because the telomere region is made up of highly This molecule has 17
nearly identical
repetitive DNA, we believe that we can use optical tweezers to detect each repeat ~200bp repeats Bockelmann, U., & et al.(1997). Molecular Stick-Slip Motion Revealed by Open-
and analyze various structures of the telomere region. These experiments could
probe G-quadruplexes, Telomerase interactions, scafolding proteins, and more.
some pictures here Mary Ann Osley ing DNA with Piconewton Forces. Physical Review Letters , 4489-4492.
Pranav Rathi
Koch, S. J., & et al. (2002). Probing Protein-DNA Interactions by Unzipping a
RNA Pol II interactions Transcription is a very complicated process Single DNA Double Helix. Biophysical Journal , 1098-1105.
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 Brian Josey Shundrovsky, A ., & et al. (2006). Probing SWI/SNF Remodeling of the Nucleo-
some by Unzipping Single DNA Molecules. Nature Structural and Molecular
that we can also unzip through Pol II during various stages of transcription for Biology , 549-554.
further insight into the process. It will also be useful to have an unzipping profile Karen Adelman Wang, M. D ., & et al. (1997). Stretching DNA with Optical Tweezers. Biophysical
when dealing with chromatin mapping in vivo.
Chromatin Mapping After Shotgun DNA Cameron Neylon Journal , 1335-1346.
Mapping, we hope to be able to map nucleosome loca-
Jean-Claude Bradley
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
Diego Ramallo KochLab
then unzipping the now naked DNA and using these force
curves to match to our database of unzipped fragments.
Stefanie Gallegos