I’ve always been curious about DNA barcoding. Interest in wides-scale DNA barcoding exercise had been around for a long time, in part due to potential for amateur scientists to contribute to cause of the sciences using relatively minimal and easily obtained equipments and reagents. There had been some high-profile events and articles involving DNA barcoding techniques applied to everyday life in recent memory, like the infamous ‘sushi-gate‘ incident. Yet how many people really know what is it and how many people have a clear understanding of how to do it? I certainly was clueless for a long time.
It’s a little weird now that I think about it. Despite doing tons of PCR reactions day-in and day-out at the Genspace lab for one reason or another, I never tried to dig into what exactly DNA barcoding entails in its visceral, barcoded details. Well, recently some of our Genspace members including yours truly went out on a sort of field trip to the Harlem DNA lab (situated within a junior high school in Harlem) for a day-long DNA barcoding workshop in preparation for the upcoming NYC Urban Barcode Project.
And the process couldn’t be easier. In a nutshell it just involved amplifying specific segments of DNA from a sample organism and sending it in to Genewiz for sequencing. The specific DNA segment to be amplified differs slightly from the kind of organism (is it a fish? Plant? Or insects?) but in case of most vertebrate mammals you use a portion of its mitochondrial genome called cytochrome c oxidase subunit I (COI) as the bacoding region. Mitochondrial genome (something I’ve been working with a lot for the past few months, ironically enough) is ideal for this sort of genetic species identification since they hit the exact sweet spot between homogeneity and differentiation within similar branches of the phyla, due to their rate of mutation and how mtDNAs are only passed through the maternal line. If you’re interested in performing your own DNA barcoding experiment outside regular lab settings or any official competition you can do so with the pdf files of requisite primer sequences already online and just order it straight from places like IDT. While specific protocols for running the PCR and prepping samples differ from place to place (I’m still looking for that perfect optimized protocol) what you are doing is a basic PCR amplification of the specific part of the mitochondrial genome, so when push comes to shove I’m sure simple chelex based DNA extraction (crush and pop in the sample with chelex beads for 10 minutes at ~99 C, centrifuge at 13000rpm for about a minute and extract the supernatant) combined with primers and PCR mastermixes or GE PCR beads (which already contain pre-made taq polymerase and buffer mixtures for optimal performance) will work just as well, provided that the sample is fresh enough. I think I’m going to run some experiments with the materials we already have at the Genspace lab and post the results later on. Once we put together a library of verified barcoding primer parts we should be able to do some very interesting projects and classes with the NYC biology community at large.
During the barcoding workshop we had a chance to pick out our own samples and run through the barcoding process with the instructors. I picked regular house ants, some random plant Ellen brought from her garden, and a YFP producing zebrafish that’s been dead for some time (it’s a long story). I went through the DNA extraction, purification, and PCR process outlined briefly above, using appropriate primers (for students participating in the competition the Dolan DNA learning center & Harlem DNA lab will provide the kits for free!). Here’s a picture of the gel we ended up with, dyed with syber-green (thanks Oliver!).
Now I seem to have misplaced the list of what each lane does, but the point is, all the barcoding amplifications worked except for the transgenic zebrafish. And it’s not just me, transgenic fish samples prepared by everyone else failed as well, something I can only attribute to the condition of the sample at the time of the barcoding experiment. You see when living things die cells lose structural integrity and rupture all over the place, mixing existing DNA molecules within the cells with all kinds of junk and nucleases that will damage the sequence. Considering the fish was stinking up to the high heavens by the time we got it to the lab that certainly sounds like a very likely scenario to me.
All the other samples works beautifully, and we prepared about 10ul aliquots of each PCR product and sent it in to Genewiz to get sequenced (the same Genewiz I got my mitochondrial DNA sequence from). They’ll be getting back to use within few days with the sequence data we can feed into public databases of DNA barcodes to determine what kind of organisms they are.
People always talk about how the field of biotechnology is advancing by leaps and bounds, and how the infrastructural developments like massive DNA sequencing centers for cheap sequencing will change how most view life and themselves. For a person not previously versed in biology like myself this was a great opportunity to come face to face with capacity for people outside of traditional academia to contribute to the sciences, using largely off-the-shelf technologies and public databases. The entire process of obtaining the sample, amplifying a specific genome within the sample, and getting it sequenced probably cost me about $5 in terms of materials. Think about that. $5 dollars to gain some level of insight into a genetic makeup of an unknown organism, open to everyone. Although this is nowhere near the kind of stuff we can do with true deep sequencing the day is coming, and it will certainly make for a very interesting world.
If you’re interested in learning more about the NYC Urban Barcode Project or DNA barcoding process in general, feel free to contact me at sung at genspace dot org. Genspace is one of the sponsors of the NYC Urban Barcode Project and we are looking forward to input and participation from students and teachers around the city!