DNA extraction and DNA Barconding

[Otto Heringer]

Hello people,

I'm planning to perform a DNA extraction to do DNA barcoding analysis with a sample of food (bovine meat, I suppose). The DNA extraction part is for a public demonstration of DIYbio and a "first experiment" introduction - one past thread on this list helped figure out this.

The objetive is do a DIY extraction using common commercial products and a "normal" extraction using standard lab reagents, just for comparison before the public demonstration.

Cathal's blog have the best source of information regarding this subject I found so far - specially where to find some "ingredents".

I'll look for genomic DNA instead of only plasmids (I'm using this protocol), but the problem of where to find some reagents persists. The only one that is hard to obtain is indeed Tris.

My plan is try to use drugs like Toradol that is usually sold with "Thrometamine", what appears to be another name for our well known Tris. I don't know if the information about the % of thrometamine on the drug will be avaliable, But I'll try do something.
Do you know other better options? Maybe a more avaliable compund that might substitute Tris?

And about barcoding, I see that there is a still open debate about the use of barcodings because of the supposition that it might "replace" taxonomy. Some say it is only useful for "biodetection" of some species and that taxonomy need more complex work.

But my point is: if barcoding is only useful for biodetection, so why is needed to use only a particular DNA region present in all species? If the biodetection was the only goal, wouldn't be better compare whole genomes and trace a map of DNA disparities between all species? Of course, taking in acount the crescent feasibility ($ and time) of whole genome sequencing.

This way just a PCR and gel would be needed - and not sequencing for every barcode sample.

I know some of you have some experience on the subject. What do you think about it?

Otto

[John Griessen]

On 01/15/2015 10:34 AM, Otto Heringer wrote:
> This way just a PCR and gel would be needed - and not sequencing for every barcode sample.
> I know some of you have some experience on the subject. What do you think about it?

No direct exp. yet, but sounds just like using a gas or liquid chromatograph output
to infer molecules in the sample. A PCR/electrophoresis only method would depend on molecular weight
and shape alone.

Would that give as much ability to discern as sequencing even a small part of a genome?
PCR/electrophoresis only might be like comparing search stats for
crime suspects by a height and body weight vs. right thumb print and shoe size.
The thumb print alone would trump
thousands of other matches based on height and body weight.

OK. Now the experienced can chime in...

[Avery louie]

Torodal is not tris.  It is a prescrition drug.

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[scoc...@gmail.com]

http://www.bostonbioproducts.com/products/chemicals/chemicals/1107-tris-ultra-pure

$30 for 500g too expensive?

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: Otto Heringer
Sent: ‎1/‎15/‎2015 11:34 AM
To: diy...@googlegroups.com
Subject: [DIYbio] DNA extraction and DNA Barconding

--

[Dakota Hamill]

DNA barcoding is pretty easy to be honest once you have the primers.  We've gotten it to work with fish, fungi, bacteria, and plants using a simple salt lysis and isopropyl alcohol precipitation method.  Follow that up with 2x Taq standard master mix, 30-32 cycles, and boom you're done.

http://www.dnabarcoding101.org/introduction.html

That honestly has everything you need.  Everything.

Genes used for barcoding are generally "agreed upon" by scientists via literature, and also at conferences.  There is now an official conference where votes are taken on which genes should be the  "standards" for different organisms.  ITS for fungi, 16s for bacteria, etc etc.  Pretty sure it's even called the barcode of life conference.

As for replacing sequencing with a gel for species ID, it will never work because they do two fundamentally different things.

The gel is going to separate your PCR products by size.  The sequencing is going to tell you exactly what the sequence is of the fragment that you amplified.  Think of a 700 base pair fragment.  Now think of how many different 700 base pair fragments there is the possibility of having with A,T,C,G.  

On a gel, those 700 bp fragments would look exactly the same because they'd run at the same rate.  On a sequencing machine you'd have a different sequence for every single one.  

I've run 12 different fungal samples before, and 11 out of 12 had a band at the same run distance.  The only thing you could get from a gel would be better resolution of the differences in the length of the amplified products.

Sometimes the ITS region is 800bp, sometimes its 600-700.  It depends on the organism.

The genes are selected because scientists have found that they have, for lack of a better term because I havn't been reading barcoding literature for a while, a balance between conserved sequences and mutation rates.  

As for above, I shouldn't say a gel could NEVER be used for species ID.  If I recall correctly, there was some insect pest that was eating a bunch of crops in south america, and they found two different species that looked almost identical, but the LENGTH of a gene fragment when PCR'd was different.  Let's say one was 100 bp larger than the other.

Turns out, one bug is "good" and one bug is "bad" but they looked almost the same from the outside.  In that case, they said they were able to simply look for the 100bp difference using the same primers and ID the good vs bad one, and forgo sequencing.  I can't remember where I read it but it was an example of how DNA barcoding helped play a big roll with treating an agricultural pest.

So yeah, 2 years ago I could have given you a more detailed answer of the who what why when and how but, that's kind of a general overview.  

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[Jeswin]

On Thu, Jan 15, 2015 at 11:34 AM, Otto Heringer <ottowh...@gmail.com> wrote:

> I'll look for genomic DNA instead of only plasmids (I'm using this
> protocol), but the problem of where to find some reagents persists. The only
> one that is hard to obtain is indeed Tris.
>

Plasmid extraction is only for bacteria. It's more difficult to do,
because we want to keep the genomic DNA from contaminating the plasmid
DNA. If you got the right reagents, its not difficult. Since you are
looking to determine beef, then you're looking for genomic DNA.

>
> And about barcoding, I see that there is a still open debate about the use
> of barcodings because of the supposition that it might "replace" taxonomy.
> Some say it is only useful for "biodetection" of some species and that
> taxonomy need more complex work.
>

You can barcode using PCR/electrophoresis if you got the correct
primer designed. Is there a sequence found in cows that is different
from one in goats, chicken, etc? With that specific sequence, you will
have an expected PCR product size. Maybe you should find more than one
highly species specific sequence. Then you can have a "fingerprint".

[Otto Heringer]

@Jhon

My hypothesis is if you can compare whole genomes, it is possible to find a set of regions that not match and that are stable enough to be considered to as a parameter to compare the different genomes. In this set is possible design primers to amplify parts of these regions and in almost an arbitrary size - I'm supposing that these regions have many different sizes, what makes this design flexible.

Whit this, just gel bands would be sufficient to say if the sample is from a species or another (just like you said, but in a different context), and the sequencing, what is needed in the case of barcodings, will not be necessary.

@Avery

I'm not referring to the Toradol by itself, but what comes with it, like this.

I didn't know that this drug need prescription. Thanks for the info.

Lets see if the prescription is needed here in Brazil too... I'll take a look.

@Sebastian

Sure, it is not so expensive. But I want use reagents that could be found in the day-to-day world of the audience. I want to bring the experiment for their context.

In short terms: I want the extraction to be the most "DIY" it can be. Just for the "show".

@Dakota

Thanks a lot for sharing your experience. Nice link, I was taking a look on this site; but I found yours better explicative.

It seems that one of the pursuits of barcoding the DNA is find a unique DNA sequence present in all (or almost all) life beings and that could be comparable - and to be comparable, it must have the balance between mutation rates and conservation, as you said.
My point is, if "barcoding is no substitute for taxonomy", so why do we need to keep trying to use the "universal" DNA piece if the uselfulness of what we call today "DNA barcode" only would be for identification of species?
If taxonomic correlations cannot rely upon DNA barcode, and taking in account the crescent viability of whole genomes sequencing, why not use little smart-chosen pieces of the thousands of non-matching sequences between genomes to identify species!? It could be possible design a set of bands of any size to confirm any species if whole genome sequencing was a trivial thing that ani DIYer could do. And in accord to the Carlson Curves, it might be so in the future.
So, my question is a little deeper: will we keep thinking on DNA barcodes as we think today if whole genome sequencing get popular!?
And, by the way, my idea is to use the known sequenced genomes to be able to identify the presence of DNA material of a suspected species "only by gel".

@Jeswin
Yeah, I'm looking for genomic DNA. I was making a comparison with the DIY extraction protocol described by Cathal on his blog.
And yes, this is exactly what I'm planning to do. Still need to do the bioinformatics. I will share here my results. Hope to find an interesting way to do this.
My intuition keeps me saying that someone already did that. But I found nothing so far. I'll appreciate if you could point anything for me take a look.

I'm even more excited about this with your replies. Thanks a lot. This is going to be very fun. :)

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[Dakota Hamill]

I wish a whole genome analysis cost as much as an 800bp Sanger read, because yes, comparing every single base pair in one organism to every single base pair in another organism would probably be the end all be all differentiation between "species".

In the end, we are all different, if only by an atom. 

As someone much more famous than I said, "Endless forms most beautiful".  Can we ever really pinpoint what exactly makes us different?  Humans are all considered to be part of the same species, but every single one of us is unique, even if we only look at the base pairs that make us up, and forget the unfathomable complexities that go along with amplifying that signal upwards to create a person with thoughts, feelings, wants, desires, hopes, dreams, and goals.  That was probably a run-on sentence.  

I love that life cannot be boxed up and bar-coded.  I love that nature has created a system in which a certain amount of uncertainty is built in.  

But I also agree that there could be more information/data points involved in taxonomy and phylogeny than just a 600bp gene fragment.  I've been looking at a boatload of bacteria recently, and morphologically they look pretty similar, so 16s DNA barcoding is one of the only tools we have available now to actually get a deeper layer of % similarity to already known species.  That should also be followed up with the fact that i don't believe anyone "checks" who submits sequence data to the NCBI.  I think there have been many examples of data being submitted under the "wrong" name, and that throws off future searches.  

If you can find something better, go for it!  

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[Matt Harbowy]

& if they won't sell to amateurs, Caisson will:

http://www.caissonlabs.com/product-T042-1KG-TRIS-Hydrochloride.php?id=920

Or Phytotech

http://www.phytotechlab.com/detail.aspx?ID=648

Matt Harbowy -hberg...@gmail.com

650 243 8467 - @hbergeronx on Twitter

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/54b82471.8587e00a.5e94.ffffeef8%40mx.google.com.

[Dakota Hamill]

Phytotech is the sponsor of the hometissueculture list-server and they're reputable and nice people, so, I'd go with them if you need tris

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/F72DC028-4518-40B6-8EE1-3172408FCB4C%40gmail.com.

[Cory Tobin]

I can vouch for Phytotech, too. Good customer support and they will
ship to residential addresses.

-cory

[Manu T]

In case you don't know with this protocole you can detect if you dna samples contain cattle, sheep, pig, goat, chicken and horse in only one run of PCR. 

[leaking pen]

the specific restriction enzymes used cut known spots that occur in pretty much all coding, and the distance between those codes, and the percentage of fragments that fit it, are actually very revealing to identify species. 

--

[scoc...@gmail.com]

If you want off the shelf pcr-able genomic DNA from meat, why not follow the Dolan DNA Learning Center's Human Cheek cell DNA extraction protocol written in the mitochondrial DNA kit they sell through Carolina Biology Supply Company. Replace proteinase K with McCormick meat tenderizer enzymes and then do an isopropyl precipitation. Using a prescription drug as a reagent is even less accessible than purchasing the reagent in the first place.

Saline, a centrifuge (hand crank? Dremel?), tenderizer, etc all fairly accessible. If you do want to make it more off the shelf why not process plants instead. Just soap, salt, and alcohol is all you need for a PCR-able crude genomic DNA extract. Meat is a little bit trickier but the tenderizer enzymes (papin proteases) will handle the tough connective tissue bits quite well. I did a science project when I was a wee lad and extracted genomic DNA from chicken liver using household products. It worked but never did any pcr on it since this was in the 1990's and PCR machines were not easily found.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

---

From: leaking pen
Sent: ‎1/‎16/‎2015 8:21 AM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] DNA extraction and DNA Barconding

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[Jeswin]

On Fri, Jan 16, 2015 at 11:05 AM, <scoc...@gmail.com> wrote:
> well. I did a science project when I was a wee lad and extracted genomic DNA
> from chicken liver using household products. It worked but never did any pcr
> on it since this was in the 1990's and PCR machines were not easily found.
>

I also think you should get the DNA from tissues like liver. I think
other organs might be too tough. Not sure though.

[Otto Heringer]

@Dakota

I'll play with the sequenced publicly avaliable genomes and see the mess that happens. I'm expetcing to get the obvious answer - "we use barcoding as we use because it is easier" - but also to learn something in the process.

@Matt and @Cory

Thanks for the recomendations!

@Manu

This is what I was expecting to someone shows up here! I should have searched for papers with this subject with another criteria. I would probably found this important article for what I want to do.

Thanks a lot for sharing this! It solves almost all the problems that I was expecting. I just need to search the same approach to identify earthworms - there's a urban legend that some fast foods use worms in their weird mix that is called "meat", haha

@Pen

Yes, but it will be needed to use restriction enzymes and if I had to choose between a restriction enzyme reaction on a whole genome and a PCR of specific regions, I choose the PCR. It is less messy to analyze the gel results.

@Sebastian

You are right. After Matt showed the companies selling those things to ppl like us (those weird creatures! #kidding) you got a better point than mine.

Thanks for the tip on the mitochondrial DNA kit. I'll take a look.

Also, I was thinking ask some chemists friends that were studing the protease activity of some tropical food (coconut, pineapple and papaya (papain!)) to give some information on the extraction viability of its proteases - I bet it is inviable, but I need to stop to speculate and search for the stuff even before asking them. It would be cooler get the proteases from the source instead from a industrialized product. :D

@Jeswin

It will probably be from the muscle (I hope so!). The sample is a crude Sfiha (I'll ask to be crude, it is not usually sold this way), an "arab mini-pizza" commonly found in some fast food companies menus here in Brazil.

Is a commom joke between my friends question the origin of the meat sfihas while eating it - something like: "Humm... *chewing*... They have put less paperboard on it this time." "Sure, this earthworm meat is fantastic today.".

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[Otto Heringer]

By the way, I never would expect this journal called "Meat Science". Kinda funny name.

[SC]

Sorry off topic, but speaking of strange journal names:  "Journal of Genetical Research" always kinda bugged me.