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.
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.
On Thu, Jan 15, 2015 at 3:34 PM, <scocioba@gmail.com> wrote:
http://www.bostonbioproducts.com/products/chemicals/chemicals/1107-tris-ultra-pure
$30 for 500g too expensive?
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Otto Heringer
Sent: 1/15/2015 11:34 AM
To: diybio@googlegroups.com
Subject: [DIYbio] DNA extraction and DNA BarcondingHello people,--I'm planning to perform a DNA extraction to do DNA barcoding analysis with a sample of food (bovine meat, I suppose). The DNA extraction part is for a public demonstration of DIYbio and a "first experiment" introduction - one past thread on this list helped figure out this.The objetive is do a DIY extraction using common commercial products and a "normal" extraction using standard lab reagents, just for comparison before the public demonstration.Cathal's blog have the best source of information regarding this subject I found so far - specially where to find some "ingredents".I'll look for genomic DNA instead of only plasmids (I'm using this protocol), but the problem of where to find some reagents persists. The only one that is hard to obtain is indeed Tris.My plan is try to use drugs like Toradol that is usually sold with "Thrometamine", what appears to be another name for our well known Tris. I don't know if the information about the % of thrometamine on the drug will be avaliable, But I'll try do something.
Do you know other better options? Maybe a more avaliable compund that might substitute Tris?And about barcoding, I see that there is a still open debate about the use of barcodings because of the supposition that it might "replace" taxonomy. Some say it is only useful for "biodetection" of some species and that taxonomy need more complex work.But my point is: if barcoding is only useful for biodetection, so why is needed to use only a particular DNA region present in all species? If the biodetection was the only goal, wouldn't be better compare whole genomes and trace a map of DNA disparities between all species? Of course, taking in acount the crescent feasibility ($ 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
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