10% Agarose and 1-2 nucleobase resolution

[Josiah Zayner]

So at Science Hack Day SF I worked with a bunch of people to attempt to perform DIY DNA sequencing without any expensive equipment or toxic chemicals.

We were not able to visualize the sequencing reactions yet(don't know if they worked). However, we were able to achieve ~1-2 nucleobase resolution in a 10% agarose gel.

Here is a blog post about it: http://doitourselfscience.blogspot.com/2014/10/science-hack-day-sf-2014-diy-dna.html

Hopefully in the next few months I plan to continue the work at Biocurious.

[SC]

It's good to test the limits of processes that you use, but FYI, a single pass sequencing reaction (~$3) is probably cheaper than a 10% agarose gel.  If this was done for R&D purposes though, then bravo.

[Josiah Zayner]

Why be so condescending? Such as the only way it will be useful is if "this was done for R&D purposes though, then bravo." That's why I don't participate in this mailing list much any more. Too many condescending people. For what?

I thought I would share an idea and technique that people could work on and develop and perhaps be the basis for simple protocols to do DNA sequencing _AT HOME_. Is this not a _D_ _I_ _Y_ Bio mailing list?

Sure I can send off my stuff to be sequenced. Though $3 is on the low side unless you are doing a ton of samples or are at a university with in house sequencing(not available to DIY people). Most sequencing I pay for at NASA is ~$5 / sample roundtrip but maybe you know of a place including shipping that I can pay $3 a sample and have same-day return, please let me know. For a single sample, I would argue that this would be almost non-existent.

The cost of running a sequencing reaction and a 10% agarose gel as calculated by the materials I purchased is only ~$3.50-$4.50 ($0.20/gram of agarose at 5 grams($1) for 50mL and 10 grams($2) for 100mL). And that is not even the cheapest materials. I paid ~$100 for 200 units of polymerase and besides agarose that was probably the most expensive thing(~$2 / sequencing reaction). I opted for a good sequencing polymerase with no 5-3 exonuclease to make sure that it wasn't something holding back the experiments.

Again, money is not the point anyways. The point is to allow people to have access to these techniques at home or in a biohackerspace. People who do DIY Bio should already have the equipment needed to run these experiments. Obviously this needs to be developed more but I wanted people to be aware so if they wanted they could help develop it more.

On Fri, Oct 10, 2014 at 2:49 PM, 'SC' via DIYbio <diy...@googlegroups.com> wrote:

It's good to test the limits of processes that you use, but FYI, a single pass sequencing reaction (~$3) is probably cheaper than a 10% agarose gel.  If this was done for R&D purposes though, then bravo.

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[Nathan McCorkle]

I've read up on this before, and even wrote up a wiki page on how I'd go about accomplishing a mini-sequencing-gel... but the basic idea was that a dye like gelRed had pretty good sensitivity, and this is based on the total number of bases available for intercalation/association.

So you start your sanger sequencing reaction with lots and lots of primer, such that each pool of amplicons is very dense, and since these co-locate during electrophoresis, you'd get a nice bright signal.

Here's the paper I was basing the general electrophoresis protocol off of, but I'd decided to try polyacrylamide since I was looking for nice-looking gel images:

http://www.biotechniques.com/multimedia/archive/00010/97225pf01_10994a.pdf

I ordered a sanger sequencing kit and got the polyacrylamide, but then got busy or something (and I don't have much biohacking community around for these projects to progress without my full commitment).... so I never tested it.

The basic math I did for the required starting primer concentration to get high enough signal density was:

nanograms_DNA_in_band = (formula_weight_of_nucleotide * length_of_DNA_in_band) * (concentration_of_limiting_reagent * microliters_of_reagent_added / num_microliters_in_liter) / num_bands_expected * num_nanograms_in_gram

nanograms_DNA_in_band needs to be larger than the dye's datasheet specs for minimum sensitivity.

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[Josiah Zayner]

Yeah, thanks Nathan. Was trying to do something a little more accessible than polyacrylamide. I mean that should totally work though. I was going to order a sequencing kit but instead decided that I might want something a little different and be able to tune my ddNTPs and such so I ordered the chemicals. This is because I think one will probably need more DNA and so instead of doing a cycle sequencing reaction of 20 cycles maybe 200 cycles(it is linear instead of exponential)?

We will see. I think this requires experimentation. I think too many people think, "Oh I can just go sequence this for $3." so no one has really looked much into this avenue or rather published on it. If you have any tips or ideas send them my way.

Thanks.

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[Nathan McCorkle]

On Fri, Oct 10, 2014 at 6:12 PM, Josiah Zayner <josiah...@gmail.com> wrote:

Yeah, thanks Nathan. Was trying to do something a little more accessible than polyacrylamide. I mean that should totally work though. I was going to order a sequencing kit but instead decided that I might want something a little different and be able to tune my ddNTPs and such so I ordered the chemicals. This is because I think one will probably need more DNA and so instead of doing a cycle sequencing reaction of 20 cycles maybe 200 cycles(it is linear instead of exponential)?

Hmm, I forgot to mention I'd ordered the terminator nucleotides separately, so for a given sample I'd use 4 lanes like radiosequencing gels, so I'd have a way to discriminate bases.

One of the things that sort of got me stuck was lack of funds when I had the time and initiative... but also the chemicals I ordered said something like 'ready to use' but didn't in fact include the polymerization accelerator. Then with being tight on money I wavered from thinking I'd wait to order more items to save on shipping, or something like that, and never ordered anything.

 

We will see. I think this requires experimentation. I think too many people think, "Oh I can just go sequence this for $3." so no one has really looked much into this avenue or rather published on it. If you have any tips or ideas send them my way.

If polyacrylamide can do single base separation (we know that is true) and that paper I sent shows dinucleotide differences on agarose, then it does seem possible to do some other stuff to push the agarose just that little bit farther. Maybe use purer agarose, or do some in-house cleanup on it (Cathal has posted some good ideas on this in the past) (probably won't beat whatever the super-expensive stuff does), do a really great job at cleaning up your DNA before mixing with the sequencing mix (phenol chloroform or a few rounds of silica adsorption follow by ethanol desalt?), I've also been wondering about pulsed-field electrophoresis as applied to this problem.

I'd think any sanger sequencing would be a minimum of $10-13 for no or minimal prep on their end, not including shipping (I don't think academic labs that are open to public/industry are even setup to mail stuff, so you'd need to drive/bus/taxi over to and from, or get a friend to do it). $3 sounds like pricing that a hospital/govt-lab would pay internally or something, but it'd have to be a huge lab for such prices. Does NASA do it on-campus or outsource or send it across the bay to LBNL/LLNL/JGI?

[Josiah Zayner]

Yeah, I mean this should totally be possible. What chemicals are you missing? Maybe I can send them your way.

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[SC]

Hi Josiah,

 

There was not a speck of anything condescending in my post.   Some people develop new techniques to save money, others develop to find new ways to do things.   I have observed some people here thinking some processes were more or less expensive than they actually are, I wasn't sure if you

may have been under the wrong impression of how much sequencing costs.  (And yes, $3/sample is per plate.)

 

I reread my post to see if there was some accidental yucky tone, but there really wasn't.

 

Sincerely, best of luck to you.

 

Stacy

 

 

[Nathan McCorkle]

On Sat, Oct 11, 2014 at 3:38 AM, Josiah Zayner <josiah...@gmail.com> wrote:

Yeah, I mean this should totally be possible. What chemicals are you missing? Maybe I can send them your way.

I think it was just the ammonium persulfate and the gelred that I needed.(gelred supposedly had better sensitivity, though their definition was loose and something like 'we can see a normal sized band in our imager with this mass ssDNA in a lane').

If you or others have the time to play with this stuff more, I'd be happy to send the sanger cycle sequencing kit and unpolymerized acrylamide to you (I could leave that out since you are already having some luck with agarose). This stuff has been in my freezer since Feb of last year, within a styrofoam cooler. I bet it's still good, but you'd want to check. I was just planning on using the included control primers and plasmid with short cycle times so extension wouldn't be too much, just so I could get better separation.

[Cathal Garvey]

DNA separates so well on agarose, and polyacrylamide is right there, so
I think you guys are right that lots of good hacks haven't been tried
due to simple lack of need.

The one that comes right to mind, for me, is semi-2D, to help with
resolving similar lanes. So, if you have two bands within a dinucleotide
of one another, they'll be indistinguishable (reliably, at least) on a
1D agarose, probably even with pulsed-field, but if you run them down
then shift sideways for a half-lane hop, you may make them more
distinct, if only to machine vision; two clusters diagonally adjacent
instead of vertically adjacent.

Just a thought, anyway; 2D is well established in proteins because of
the colocation problem, but has never been called for in biology. In
DIYbio, where acrylamide is less desirable as a method, there might be
room for 2D?

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[CodonAUG]

200 cycles probably won't work because you will have a really poor signal/noise ratio.  It would be better to do a 20 cycle reaction, take about 1 uL of this 1st reaction and run another 20 cycle reaction (still going to be getting more noise, but it shouldn't be as bad as 200 cycles in a row).