This is my poster presentation from the annual Biophysical Society Meeting in San Francisco, CA. I detail the current progress made in Shotgun DNA mapping and include an aside about open notebook science and my scientific life on the internet.
New High Throughput Sequencing technologies at the Norwegian Sequencing Centr...Lex Nederbragt
A talk I gave at the Microbiology Research Group (University of Oslo) about new High Throughput Sequencing instruments at the Norwegian Sequencing Centre. I also mentioned future upgrades, and the upcoming nanopore sequencing platform of Oxford nanopore
New High Throughput Sequencing technologies at the Norwegian Sequencing Centr...Lex Nederbragt
A talk I gave at the Microbiology Research Group (University of Oslo) about new High Throughput Sequencing instruments at the Norwegian Sequencing Centre. I also mentioned future upgrades, and the upcoming nanopore sequencing platform of Oxford nanopore
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
Providing tools that insure excellent Cell Based Assays is a cornerstone of our business strategy. Lauren McGillicuddy and her team at Essen Bioscience have been using our E18 Primary Rat Cortical Neurons to develop NeuroTrakTM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayerTM assays that can be run in IncuCyte ZoomTM.
This is a compilation of the Yeast genome project from the different databases and sources.
By:
Nazish Nehal,
M. Tech (Biotechnology),
University School of Biotechnology (USBT),
Guru Gobind Singh Indraprastha University (GGSIPU),
New Delhi (INDIA)
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
Providing tools that insure excellent Cell Based Assays is a cornerstone of our business strategy. Lauren McGillicuddy and her team at Essen Bioscience have been using our E18 Primary Rat Cortical Neurons to develop NeuroTrakTM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayerTM assays that can be run in IncuCyte ZoomTM.
This is a compilation of the Yeast genome project from the different databases and sources.
By:
Nazish Nehal,
M. Tech (Biotechnology),
University School of Biotechnology (USBT),
Guru Gobind Singh Indraprastha University (GGSIPU),
New Delhi (INDIA)
This is a presentation I gave for my Candidacy for PhD. I present on the possibilities of probing protein-DNA interactions using Optical Tweezers. I discuss simulating force curves from optical tweezers, background information, and the molecular biological preparations involved. Finally I conclude with future applications of the technique that range from analysis of alternative splicing, transcriptional studies, and telomere mapping.
A practice talk about Shotgun DNA Mapping. I talk about gene expression, optical tweezers, unzipping DNA, mapping DNA fragments, telomeres, RNA transcription, and much more!
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.
I made this presentation to show some collaborators whose lab I worked in last semester. In it I discuss the past, present, and future of Shotgun DNA Mapping and all that it contains.
To describe DNA extraction
To explain and demonstrate DNA cloning
To explain the process of PCR and its uses.
To explain DNA fingerprinting and its uses
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes.
This chapter provides an overview of DNA microarrays. Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. We first cover the history of microarrays and the antecedent technologies that led to their development. We then discuss the methods of manufacture of microarrays and the most common biological applications. The chapter ends with a brief discussion of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
What is blotting? Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.
DNA fingerprinting is a laboratory technique for determining a person's likely identity based on the nucleotide sequences of specific regions of human DNA that are unique to individuals.
The above presentation consist of the definition of microarray, brief history, general principle of the same, the type of scanner that are used to read or to scan the microarray , type of DNA microarray and finally its various apliccation including the role of DNA microaarray in drug discovery.
can someone explain in a easy way chromosome walking I know its w.pdfmckenziecast21211
can someone explain in a easy way chromosome walking? I know it\'s with overlapping
fragments and a gen nearby the gen you want to find, but I don\'t clearly understand it, I have
images in my book, but an image with steps and explanation would be nice,
Solution
Chromosome Walking:
Chromosome walking is positional cloning of gene sequences or upstream promoter sequences.
Eukaryotic DNA contains a lot of junk DNA between the genes. The technique relies upon
screening of gDNA library for overlapping fragments in a sequential manner to tap the upstream
or downstream region.
Procedure:
1. Isolation of Genomic DNA: First of all isolate genomic DNA (gDNA) of the test organism
and then partially digest it with some restriction enzyme, say Hind III that would yield unequal
random fragments.
2. Cloning of the Digested into E.coli: Clone the digested gDNA into a cloning vector (between
the Hind III sites in this case) to prepare a gDNA library of the organism. Each colony of the
library would have only one clone.
3. Preparation of marker probe: Prepare a radioactively labelled probe from the neighboring gene
(say A), from where you want to start walking.
4. Colony Hybridization: Hybridize the probe with your gDNA library. The probe would show
the signal in the colonies carrying DNA in and around your gene.
5. Sequencing: Pick the colonies showing the signal and isolate the plasmid DNA and sequence
each using the plasmid-specific primers. You would get only one sequence per bacterial colony
of your library.
The sequence, thus obtained must be analyzed. Select longer sequences towards the direction of
your walk.
6. Preparation of the probe: Prepare new probes from the region towards your target gene.
7. Repeat the steps 4-6 till you get the clone and sequence of your target gene..
The Modern Cuisine Movement: The Intersection of Science and Culinary ArtsAnthony Salvagno
In 1988, molecular gastronomy was born. The field is aimed to investigate the scientific processes of cooking by: (1) the social phenomena linked to culinary activity, (2) the artistic component of culinary activity, and (3) the technical component of culinary activity.
This talk was hosted at Explora! on 11/20/15 for their Science of Food event. This presentation and demonstration was funded through Girl Scouts of New Mexico Trails
“Open source” is the act of providing software, technology, or process for free and inviting amendments and derivation. Open sourcing philosophy has emerged primarily in the software industry, but over the past decade the open movement has risen from an unconventional concept to an emerging approach now applied to many other fields including communication, STEM, art, and many others. When money is involved, open policies are seen as unfavorable and have been largely disregarded. Current resistance to open access is not well understood but may be stem from questions of profitability or fears of losing intellectual property. In this open discussion, which we want the audience to participate in, we hope to present the possibility of using open practices to build a successful business model. What if providing open access could make a company more profitable? What if transparency brought more customers? What’s more, what if open practices could add a new dimension to corporate social responsibility? We present the idea that openness in a corporation would actually be extremely beneficial to businesses and organizations alike, and could drive profitability and induce trust while minimizing corruption and ensuring corporate, societal, and fiscal responsibility.
Deuterium is present in all naturally occurring water, and accounts for 1 in 6420 hydrogen atoms. Seemingly insignificant, that ratio is actually quite impactful in life sciences. Pure deuterium oxide (heavy water) has been found to be toxic to eucharyotic organsims, and has been found to be inhibitory in smaller doses. Here I present results that demonstrate that in even smaller doses the concentration of deuterium has a dramatic effect on life processes. I also show that at the molecular level, deuterium has a stabilizing effect. By simply changing the properties of the solvent, there can be dramatic effects that impact a variety of experiments.
For the entire story, please refer to my dissertation: https://docs.google.com/document/d/1p9-qqF8oUcwNGJjZG3CoKL2xvIhbqKCFQ7_faegNPIU/edit?usp=sharing
Presented on 2/15/13 for Student Research Day at UNM. Scientific information is published in peer reviewed journals as a complete account. Unfortunately there is nothing complete about the process. Frequently intermediate and supplementary experimental data is excluded, methods are not nearly detailed enough, and results are manipulated for appearance. Open notebook science aims to be a complete account of the scientific process: from ideas and planning, to experimental setup and protocol design, all the way through data interpretation and commentary. Here I
talk about tools to create and support an open notebook, effective use of the medium, and how social interaction creates a community using examples from my own open notebook for research.
Here I present some of the best tools for open notebook science and a brief discourse as to what open notebook science is, why it is important, and how it will permeate scientific culture.
My abstract from SACNAS:
Open Notebook Science - UNM Biomed Symposium EditionAnthony Salvagno
Scientific information is published in peer reviewed journals as a complete account. Unfortunately there is nothing complete about the process. Frequently intermediate and supplementary experimental data is excluded, methods are not nearly detailed enough, and results are manipulated for appearance. Open notebook science aims to be a complete account of the scientific process: from ideas and planning, to experimental setup and protocol design, all the way through data interpretation and commentary. Here I talk about tools to create and support an open notebook, effective use of the medium, and how social interaction creates a community using examples from my own open notebook for research.
A presentation I gave to students at the New Mexico Graduate and Professional Student Conference. Open science is the future of science, and open notebook science is the future of how scientific information is accessed and propagated. Here I present examples from my notebook and from a case study of an open notebook community (Physics 308L Junior Lab).
Here is a summary by Andy Maloney and myself. This describes what we basically do in the lab and why we have so much fun doing it. This presentation was a blast to make and I look forward to doing many more.
Credit: The song is "Missing You" by Trash80 from the album Icarus. It can be downloaded from 8bitpeoples.com.
This is the presentation based on our propsal of the same name. The presenters were Laura Pawlikowski, John Montoya, Ken Seal, and myself. We discuss functionalizing an AFM tip with antibodies for protein detection and discuss several possible uses for such a device. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins).
Proposal for Protein-DNA Mapping using AFM for Lab on a ChipAnthony Salvagno
This was a report written for a class I am taking in conjunction with Ken Seal, John Montoya, and Laura Pawlikowski. We discuss functionalizing an AFM tip with antibodies for protein detection. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins). This proposal details everything we imagined. We presented on this topic as well.
My girlfriend made a presentation summarizing her REU experience. It is about gravitational lensing and the work she did trying to distinguish characteristics of lenses caused by galactic clusters. That is the best summary I can give.
The Biological Preparation Of Shotgun DNA Mapping 5/15/09Anthony Salvagno
This is a talk I gave at the 1st KochLab Symposium. This talk provides a glimpse into my work at Osley Lab during the Spring Semester of 2009 at UNM. I present making random genomic fragments, cloning those fragments, and ligating the fragments to a construct that enables said fragments to be unzipped using an Optical Tweezer.
This was a practice talk I gave in front of the IGERT people at UNM. It is a brief discussion of the things I do in KochLab up to this point (Summer 2008).
This is a talk I gave at the 1st KochLab Symposium. I briefly provide a crash course in genetics and how it relates to our research. I specifically talk about trascription, translation, DNA replication, restriction enzymes, plasmids, and some applications of all.
BPS 2010 Poster Presentation: Shotgun DNA Mapping with Yeast
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