Alternatives to Gel Electrophoresis
Tito Jankowski
There are a few tried-and-true tools of the DIY biologist toolkit -
gel electrophoresis being one of them. We use gels to separate pieces
of DNA by length - this might be more familiar to those of you who
have checked out the DIYbio blog
(http://blog.diybio.org/2008/07/diybio-3-gel-electrophoresis.html)
However, this technology is 30 years old - how can newer technology
help us separate pieces of DNA by length?
I've talked with Tom Knight a bit about one alternative: capillary gel
electrophoresis. Rather than working with a slab of DNA, you can
imagine this as running a gel through a straw. It's a more refined
procedure, faster, and easier to take readings off of than traditional
gel electrophoresis. However, according to my research, it has been
applied only to DNA sequencing. To pursue this idea for DIY use would
take a good bit of hacking :)
Do any DIYers have experience with capillary electrophoresis?
How else can we separate pieces of DNA by length?
Best,
Tito
Bryan Bishop
> However, this technology is 30 years old - how can newer technology
> help us separate pieces of DNA by length?
Specifically, let's write up a few requirements:
* No nasty chemicals, preferably no gels
* Simple -- straws are ridiculously simple and a good example
* Reasonable time frames of operation
* Eyeable visibile separation information
* .. anything else?
> Do any DIYers have experience with capillary electrophoresis?
Links?
> How else can we separate pieces of DNA by length?
I recall posting here in July about some other methods .. but let me
append some more that I've been thinking about:
> -It would be nice if we could hack the gels so that they could be
> used more than once.
Awesome idea, yes, definitely. Have you seen the suggestions to use
artificial gels? The latest I remember hearing of was the silicon gel.
"Entropic trapping and sieving of DNA in nanofluidic channels"
http://www.hgc.cornell.edu/biofab/entropic.htm
http://www.hgc.cornell.edu/biofab/gel.htm
> The first result of lambda DNA movement in this channel shows the
> possibility of a new kind of sieving device for polymers and
> macromolecules. In a flow of very low concentration of DNA,
> individual DNA molecules were retarded by the entropic barriers posed
> by the interface between thin and thick region of channel. Below a
> certain driving electric field, this effect decreases the mobility of
> DNA drastically, suggesting a trapping of DNA molecule.
>
> Recent experimental results shows that there is actually a difference
> in mobility between larger and smaller DNA molecules. In contrary to
> our intuition from conventional gel electrophoresis, larger DNA
> molecules turned out to move faster than smaller ones in this
> channel.[3] This is due to the fact that DNA molecules are heavily
> deformed and stretched when they enter the thin gap, and this
> deformation energy barrier, not the entropic free energy difference
> between spherical and compressed molecule, is the relevant energy
> barrier in the escape of DNA molecule.[4]
>
> This separation device is a very promising candidate for an
> alternative of gel electrophoresis, Several advantages over gel
> electrophoresis or other newely emerging technologies include, (1) It
> is not a time-consuming pulsed-field technique but requires only a dc
> field to control the device. (2) It is peculiar because one can
> recover the longer DNA molecules first, in contrast with the gel
> electrophoresis where longer molecules are generally 'stuck' at the
> first part of the gel. (3) It is very easy to control the gap size
> (or etch depth) and there is no practical limit in terms of how
> narrow they can be made. Therefore one can easily optimize the device
> for a desired length range of DNA for an efficient separation. (4)
> Since we are making use of only z-directional size constriction, we
> could make a large area of this structure on a Si wafer for a
> paralleled operation of many samples.
>
> To see a video clip of the DNA molecules moving in the channel, click
> here. This page will load two large avi files which requires
> appropriate plug-in to see.
http://news.bio-medicine.org/biology-news-2/Artificial-Gels-Could-Speed-DNA-Sequencing-13341-1/
> Stephen Turner, a graduate student working under Harold Craighead,
> Cornell professor of applied and engineering physics, described his
> biochip research in a talk, "DNA Motion in Nanofabricated artificial
> Gels," today (March 25) at the centennial meeting of the American
> Physical Society in the Georgia World Congress Center.
<snip>
> replacing the organic gel with a tiny solid-state device, called an
> artificial gel. electrophoresis gels consist of a maze of
> interlocking polymer molecules that leave many tiny openings through
> which moving DNA molecules must navigate. Using the same techniques
> used to make electronic circuits, tiny passageways can be carved on a
> silicon chip.
>
> Turner's artificial gels are forests of vertical pillars with sizes
> down to 100 nanometers (nm) thick and 100 nm apart. (A nanometer is
> one billionth of a meter.) They are smaller, Craighead believes, than
> earlier versions of artificial sieves, an achievement made possible
> by using the Cornell Nanofabrication Facility's electron-beam
> lithography tools, which can lay out features much smaller than those
> used so far in commercial integrated circuits. Ordinarily such
> devices are made by etching a cavity in the silicon, then gluing on a
> cover to create a channel through which the DNA sample can flow.
> Turner used a new technique in which the channel is filled with a
> "sacrificial layer" that can be etched out after a covering layer is
> deposited. This allows much more precise control of the height of the
> channel, he explained.
>
> The researchers are still at an early stage, running DNA samples
> through the biochip to see how fragments of different lengths can be
> identified. They mount the chips between two microscope slides, glue
> small reservoirs to each end to hold a few drops of a water-DNA
> mixture, place the slides on a microscope stage and apply an electric
> field, then watch and measure what happens, tagging DNA molecules
> with fluorescent dyes to make them visible.
So that's one option. The fabrication of these artificial gels is rather
intense, with the nanolithography and nanofabricational setup required
for it. It's just one alternative. I am sure there might be others.
DNA electrophoresis in microfabricated arrays:
http://www.nano.umn.edu/research_projects/2007/DNA_Electrophoresis.pdf
Was that it? Hm. This one might be the one I'm thinking of:
http://web.archive.org/web/20020909121552/http://polymer.matscieng.sunysb.edu/dina01/
> Performing electrophoresis on a flat silicon chip, introduced in this
> research paper, promises to alleviate these drawbacks, allowing for
> fast, efficient, automated results of high resolution quality.
> Electrophoresis was performed using 1 kb Ladder DNA with a high
> resolution of 300 bp at a relatively low electric field of 4.5 V/cm
> (This is comparable to capillary electrophoretic performance at much
> higher fields >100 V/cm). A double-logarithmic plot of mobility of
> the DNA chains (μ) vs. number of base pairs (N) shows that there is
> indeed a length-dependent mobility when performing electrophoresis on
> a chip.
Anyway, there was a group that had a silicon surface that you could
electrically drag DNA molecules through and get some serious results.
The staining was typical, but it wasn't a gel.
Now some new stuff: there was recently a few papers out there about
shrinky dinks and 3D microfluidics baking in conventional ovens. I have
to admit that I am a little confused about the claims that these papers
are making though; they claim that these shrinky dink microfluidic
circuits can be used to detect various antibodies, but I don't see how
without incorporating some sensors into the shrinkydinks. So if
somebody can figure out how to actually use fluidic logic gates in
these shrinkydinks then maybe there's some possible DNA separation that
can be done. And nearly everyone has access to an oven. If not, a
really hot toaster oven could be built.
Probably a few other ideas I'm forgetting at the moment.
- Bryan
________________________________________
http://heybryan.org/
Engineers: http://heybryan.org/exp.html
irc.freenode.net #hplusroadmap
Jim H
Yes, we used CE for QC of large oligos at IVGN/LTI. Here's a good
review article (warning PDF): http://www.cstl.nist.gov/strbase/pub_pres/Butler2004a.pdf.
Essentially, PCR to amplify target and tag DNA fluorescently, separate
on CE column, detect using lasers, software to interpret data.
For standard, short primers much higher throughput method is Mass
Spec. That one's even harder to hack!
On Oct 19, 10:08 am, Bryan Bishop <kanz...@gmail.com> wrote:
> On Saturday 18 October 2008, Tito Jankowski wrote:
>
> > However, this technology is 30 years old - how can newer technology
> > help us separate pieces of DNA by length?
>
> Specifically, let's write up a few requirements:
> * No nasty chemicals, preferably no gels
> * Simple -- straws are ridiculously simple and a good example
> * Reasonable time frames of operation
> * Eyeable visibile separation information
> * .. anything else?
>
> > Do any DIYers have experience with capillary electrophoresis?
>
> Links?
>
> > How else can we separate pieces of DNA by length?
>
> I recall posting here in July about some other methods .. but let me
> append some more that I've been thinking about:
>
> > -It would be nice if we could hack the gels so that they could be
> > used more than once.
>
> Awesome idea, yes, definitely. Have you seen the suggestions to use
> artificial gels? The latest I remember hearing of was the silicon gel.
>
Kay Aull
capillary electrophoresis.
<infodump>
In my day job, I use this one from Caliper:
http://www.caliperls.com/products/labchip-systems/labchip90.htm
It has a little capillary tube, about the dimensions of a syringe
needle, that sips up sample from your 96/384 well plate into a
detector. It covers the size range of standard agarose gels, with
about the same resolution, and takes ~30 sec per sample. We rely on
it for QC'ing the DNA we build, and it works well. They say the
machine does RNA and protein too...never tried RNA, but the protein
detection is unreliable (in my experience).
This method is, basically, a scaled down gel. The chip is primed with
an acrylamide/ethidium bromide mix, which the DNA is pushed through
under high voltage. (Which is highly non-ideal from the DIY safety
angle, but...oh yeah, there's a UV laser in there too.) IIRC plain
capillary electrophoresis doesn't do well on DNA, because the larger
polymers have increased size, and thus drag, to balance out the
increased charge.
While it might not be impossible to homebrew one of these puppies -
heck, there are people building fusion reactors in their garages - I'd
still be extremely impressed. It wouldn't be the poor DIYer's gel
substitute, that's for certain.
There are also microfluidic strategies for doing electrophoresis.
Some labs have built chips in which the DNA has to navigate through
microfabricated posts - this is the same principle as gel separation,
except the obstacles are of a set size instead of the random
distribution found in a gel.
There's also the entropic sieve method. For this one, you need a
series of deeper "wells", separated by narrow channels. When DNA is
in its highest entropy state, it curls up into a ball - longer DNA
makes a larger ball. In the wells, it has room to relax into this
state. The longer a DNA molecule is, the less likely it will un-ball
itself so that it is narrow enough to pass through the channel. Thus
longer DNA moves more slowly through the series of wells.
I don't know how practical any of this microfluidics stuff is for home
use - never seriously scoped it out. Fabrication could be outsourced,
and plenty of labs do, but I'd be curious about the price (even if the
chips could be reused...plus, you need the equipment to run them). My
understanding is, also, that these methods are largely in alpha. So
user beware.
On the cheap n' dirty side, if you just want to see DNA, you can play
with dsDNA-specific dyes like SYBR Green. I've tried using it for
colony PCR in my own work, to avoid having to run a gel - it can be
done, though I'm not sure I'd recommend it (needs more development
work before I'd inflict that protocol on someone else).
</infodump>
- Kay
Tito Jankowski
30 seconds a run is pretty classy - Glad to hear it works!
The post/well chips are interesting too - I emailed each of the Profs
to get the latest word on those projects. I'll keep you posted, though
I agree these tools are still in the experimental stage.
Jim - how long were the oligos you ran? With your experience, does
this protocol have DIY potential for sequences of a few kb?
Overall:
What if we didn't need fancy nano-posts and wells, but simply a common
material which is complex enough at the DNA scale? After all, agar is
just a mush of seaweed extract. Does anything come to mind?
Tito
Norman
30 second separation would be nice... and ability to separate and use
the dna for downstream (more than just visualization) without gel
cutting and purification would be a good advance in technology even
for a full-scale molecular biology lab. Gel cutting and purification
is one of the biggest road blocks in our BioBrick assembly protocol,
we end up loosing a large percent of our DNA and must start with huge
plasmid preps. The expensive gel purification kits (with the silica
filter) hasn't worked as advertised for us, on a good day we are lucky
to get 10% DNA back.
I've been trying to hack something together similar to the clonewell
by Invitrogen. That ended up wasting lots of time because you need to
wait for each band to pass the second row of wells. However you can
use propanol/ethanol (commonly available reagent) precipitation
without the gel purification kits to concentrate the DNA from the
liquid picked up. If you load enough DNA (lots!) on gel, it's visibly
stained using SYBR Green in plain light, it appears as a faint pink
band.
Regarding using seaweed, we (accidently) tried agar-agar and the
resolution is not as nice as using purified agarose. It came out as a
blurred twin-band for every band on agarose.
Tito, were you at SB4.0?
Norman
---
Molecular Biosciences and Bioengineering
University Of Hawaii Graduate Program
On Oct 19, 9:07 pm, "Tito Jankowski" <titojankow...@gmail.com> wrote:
> Kay - That Caliper robot is nice, though it needs to be pocket sized.
> 30 seconds a run is pretty classy - Glad to hear it works!
>
> The post/well chips are interesting too - I emailed each of the Profs
> to get the latest word on those projects. I'll keep you posted, though
> I agree these tools are still in the experimental stage.
>
> Jim - how long were the oligos you ran? With your experience, does
> this protocol have DIY potential for sequences of a few kb?
>
> Overall:
> What if we didn't need fancy nano-posts and wells, but simply a common
> material which is complex enough at the DNA scale? After all, agar is
> just a mush of seaweed extract. Does anything come to mind?
>
> Tito
>
Jim H
Oligos up to 40 mer, I think we'd make them up to 60 mer on a custom
basis. Typically they are less than 10 bases.
CE was a custom QC for a few industrial customers.
The hardest part to hack would be the detection. One way to hack it
might be to fractionate the eluate (like chromotagraphy), then use a
spectrophotometer. I suspect this might be difficult because the
volumes are a lot smaller and concentration low for UV detection, plus
add a UV spec doesn't exactly make it easier..
On Oct 20, 3:07 am, "Tito Jankowski" <titojankow...@gmail.com> wrote:
> Kay - That Caliper robot is nice, though it needs to be pocket sized.
> 30 seconds a run is pretty classy - Glad to hear it works!
>
> The post/well chips are interesting too - I emailed each of the Profs
> to get the latest word on those projects. I'll keep you posted, though
> I agree these tools are still in the experimental stage.
>
> Jim - how long were the oligos you ran? With your experience, does
> this protocol have DIY potential for sequences of a few kb?
>
> Overall:
> What if we didn't need fancy nano-posts and wells, but simply a common
> material which is complex enough at the DNA scale? After all, agar is
> just a mush of seaweed extract. Does anything come to mind?
>
> Tito
>
Bryan Bishop
> There are also microfluidic strategies for doing electrophoresis.
> Some labs have built chips in which the DNA has to navigate through
> microfabricated posts - this is the same principle as gel separation,
> except the obstacles are of a set size instead of the random
> distribution found in a gel.
>
> There's also the entropic sieve method. For this one, you need a
> series of deeper "wells", separated by narrow channels. When DNA is
> in its highest entropy state, it curls up into a ball - longer DNA
> makes a larger ball. In the wells, it has room to relax into this
> state. The longer a DNA molecule is, the less likely it will un-ball
> itself so that it is narrow enough to pass through the channel. Thus
> longer DNA moves more slowly through the series of wells.
>
> I don't know how practical any of this microfluidics stuff is for
> home use - never seriously scoped it out. Fabrication could be
> outsourced, and plenty of labs do, but I'd be curious about the price
> (even if the chips could be reused...plus, you need the equipment to
> run them). My understanding is, also, that these methods are largely
> in alpha. So user beware.
Shrinky-Dink microfluidics: rapid generation of deep and rounded
patterns
http://heybryan.org/~bbishop/docs/Shrinky-Dink%20microfluidics:
%20rapid%20generation%20of%20deep%20and%20rounded%20patterns.pdf
> We present a rapid and non-photolithographic approach to microfluidic
> pattern generation by leveraging the inherent shrinkage properties of
> biaxially oriented polystyrene thermoplastic sheets. This novel
> approach yields channels deep enough for mammalian cell assays, with
> demonstrated heights up to 80 mm. Moreover, we can consistently and
> easily achieve rounded channels, multi-height channels, and channels
> as thin as 65 mm in width. Finally, we demonstrate the utility of
> this simple microfabrication approach by fabricating a functional
> gradient generator. The whole process—from device design conception
> to working device—can be completed within minutes.
Guess this is only for ridiculously long strands of DNA. Okay. Maybe
not. But with that sort of turn around it's worth investigating.
- Bryan
________________________________________
Jim H
machine (older model probably form the 90's) into the dumpster. We
could tell the power supply was shot, but could've hacked out the
lasers and other stuff. We had it for over a year and it was sitting
in someone else's space and they wanted us to clean it up. And there
was a flow cytometer in there, too....
We got them for "free" while acquiring a "lot" of stuff from a company
that went under. They said for $1500 we could take away everything as
long as it was gone by the end of the week.
Next time I'll put it in the mail for you....
JonathanCline
(does it work?)
DNA Sequencing and Separation in Free Solution Using Engineered Drag-
Tags
End-Labeled Free-Solution Electrophoresis (ELFSE) is a strategy for
size-based separation of DNA in free solution that promises improved
performance while avoiding the fundamental and practical limitations
associated with electrophoresis in a gel or sieving matrix. In ELFSE,
each DNA molecule in a mixture is conjugated to a polymeric "drag-tag"
that modifies the free-draining properties of the DNA, enabling
electrophoretic separation without a polymer matrix. Very rapid
separations with long read lengths are theoretically possible using
ELFSE. Since no viscous sieving matrix is necessary, ELFSE should
simplify the transition of DNA sequencing separations onto integrated
microfluidic devices currently under development.
The primary obstacle to competitive sequencing with ELFSE has
historically been the availability of drag-tags that are sufficiently
large and monodisperse for high-resolution separation of a wide range
of sizes of DNA. .....
## JonathanCline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################
Tito Jankowski
That sounds really cool, have you found anyone successfully putting
ELFSE to use?
Tito
Mackenzie Cowell
Mac
Mackenzie Cowell
2. ...I think having an easy, cheap, safe, and reliable miniprep protocol is more important than reinventing the gel electrophoresis wheel. Making competent cells with the same constraints would also be great. But what organism should we work with?
3. As Reshma's mentioned in a different thread, E. coli is going to be problematic from a PR perspective, simply because it is a household phrase associated with illness. However, E. coli k-12 strains and derivatives are pretty much exempt from the NIH Recombinant DNA Guidelines, which suggests to me that some academics and regulators may be ameniable to the idea of using it in a DIY setting.
I'm working through the review above. If I can get through it, I'll try and post a summary of what I've learned.
Mac
Tom Knight
http://nar.oxfordjournals.org/cgi/content/full/32/19/5780
Mackenzie Cowell
Quotes:
1. ADP1 is naturally competent: "...these properties allow genetic manipulation by simple addition of linear PCR products to small volumes of growing cell culture, followed by a few hours of incubation and plating on appropriate selective media"
2. ADP1 is similar to E. coli: "...the close relationship between E.coli and ADP1, combined with the newly available whole-genome sequence of ADP1, allows the tremendous amount of existing knowledge related to gene function and metabolism of E.coli to be applied directly to ADP1
How would biobrick alpha parts work with ADP1?:
1. Would one just linearize a biobrick part + plasmid and hope it self-ligated inside ADP1 after uptake? Do circular vectors even exist in Acinetobacter?
2. Or would we be trying to integrate the biobrick part into the ADP1 genome with homologous recombination? If so, what might a modified biobrick transformation strategy look like for ADP1
3. Lastly, do you think we would need to do any codon optimization when transferring E. coli biobricks (or any other construct) into ADP1?
Thanks a bunch TK!
Money quote:
Meredith L. Patterson
> 1. Would one just linearize a biobrick part + plasmid and hope it
> self-ligated inside ADP1 after uptake? Do circular vectors even exist in
> Acinetobacter?
"The first advantage of Acinetobacter ADP1 is the property of natural
competence (8), which extends to both plasmid DNA and linear
fragments."
-- Metzgar et al. _Nucleic Acids Research_ 32 (19): 5780.
http://nar.oxfordjournals.org/cgi/content/full/32/19/5780
--mlp
Tito Jankowski
but it didn't work very well for us last summer, I would love to hear
that it works well for others.
Tito
Jason Kelly
Also, there is a prof at Emory (Ichiro Matsumura) who has been
building out BioBrick parts for Acinetobacter, though when I talked to
him last they hadn't yet been submitted to the registry.
I asked him about it as a model org awhile back, one thing he said to
watch out for is that it's somewhat genetically unstable. (i.e.. high
mutation rate) However, I think that might be an OK tradeoff if it's
really as easy to work with as described.
thanks,
jason
Mackenzie Cowell
How would you recommend contacting Prof. Matsumura about getting an ADP1 strain from him and some of his ADP1 biobricks?
Maybe this question is premature. Let me ask a more timely question.
We are slowly spinning up a lab with the NUB folks - What is the proper "due diligence" concerning safety practices and regulations in a DIY context?
I'm starting by reading through the NIH guidelines and meeting with some Environment and Health Safety (EHS) officers from local institutions to ask them the same question.
Mac
Bryan Bishop
> I'm starting by reading through the NIH guidelines and meeting with
> some Environment and Health Safety (EHS) officers from local
> institutions to ask them the same question.
Mind linking to the guidelines?
- Bryan
________________________________________
Julie E Norville
listed below.
Julie
Before submitting an order you will be asked to read and accept the terms and
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products.
paper that says the atcc number
http://aem.asm.org/cgi/reprint/72/1/932.pdf
http://nar.oxfordjournals.org/cgi/content/full/32/19/5780
paper that talks about transformations
ADP1/BD413b Soil 14 x 7.7 AY289925
Print this Page
Bacteria
ATCC® Number: 33305? Price: $34.00
Preceptrol® Culture
Organism: Acinetobacter baylyi deposited as Acinetobacter
calcoaceticus(Beijerinck) Baumann et al.
Designations: BD413 [Str s] Isolation: Unencapsulated mutant derived from
ATCC 33304
Depositor: E Juni
Biosafety Level: 1 Shipped: freeze-dried
Growth Conditions: ATCC medium 44: Brain heart infusion agar or brain heart
infusion
Temperature: 37.0°C
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Anyone purchasing ATCC material is ultimately responsible for obtaining the
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Related Products
Cross References: Nucleotide (GenBank) : U77680 Acinetobacter calcoaceticus
fatty acyl-CoA reductase (acr1) gene, partial cds.
Comments: transformation [7569]
Gene mapping [9354]
Listed incorrectly as ATCC 33505 in Gilstrap et al., Experiments in
Microbiology, 2nd edition
References: 7569: Juni E, Janik A. Transformation of Acinetobacter
calco-aceticus (Bacterium anitratum). J. Bacteriol. 98: 281-288, 1969. PubMed:
5781579
9354: Sawula RV, Crawford IP. Mapping of the tryptophan genes of Acinetobacter
calcoaceticus by transformation. J. Bacteriol. 112: 797-805, 1972. PubMed:
5086660
28184: Reiser S, Somerville C. Isolation of mutants of Acinetobacter
calcoaceticus deficient in wax ester synthesis and complementation of one
mutation with a gene encoding a fatty acyl coenzyme A reductase. J. Bacteriol.
179: 2969-2975, 1997. PubMed: 9139916
Norman
person undergraduate team. Having to keep track of what antibiotic is
in what can get messy when transforming/culturing things in a large
parallel assembly-line workflow. Also, the antibiotic modes of action
are not equivalent. Personally I advise the undergraduates to stick
to Ampicillin/ccdB selection to keep thing simple.
Norman
On Oct 21, 6:38 pm, "Tito Jankowski" <titojankow...@gmail.com> wrote:
> Good idea, Mac! Does anyone here swear by 3A ligation? I love the idea
> but it didn't work very well for us last summer, I would love to hear
> that it works well for others.
>
> Tito
>
> > Norman, have you investigated 3-antibiotic assembly? It works around the
> > gel purification step.
> >http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly.
> > Cheers!
>
> > Mac
>