Construction site for "Barcode of life" project
A wiki for building our collaborative description of our project on species identification using DNA "barcoding"
Introduction (from Will 4/26)
On this planet there exist millions of different species. There are many that a child could tell apart without having to look twice, others however take much more specialized training to identify. Using genetic sequencing technologies we can identify with greater accuracy and speed potentially unknown species or help to track migrations of those kown around the world.
Barcode of Life is a global initiative to help catalogue the many different types of life this planet has to offer. Using specific genetic markers that all creatures carry, we can accurately identify shed organic material from millions of different species. By using sequencing techniques such as mitochondrial DNA extraction, we can plug in the HVS1 information into different search engines, such as the National Center for Biotechnology Information (NCBI) and with a few clicks know the organisms hertiage and potential origins. We have the ability to collect animal scat, extract genetic material, sequence it and enter the resulting information into a database and quickly be rewarded with several different potential matches or the originating organism. Then, depending on what we already know about the sample, we can, with some degree of certainty, declare what we believe the species to be.
This technology allows for scientists around the world to help better classify and categorize species living in different habitats. The benefits afforded to science allows even amateurs to go out and explore their natural world, helping to potentially identify local species, maybe even finding a new one along the way.
To begin the process our class needed to collect DNA samples to use as a base for our experiments. We decided on the hogback conservation area as our place of collection as it was nearby and we were likely to find interesting samples. Once there, we split up and stored any sign of possible animal matter in the woods and on the trail leading up Mount Olga. We collected only animal samples because we could use one primer for all of them during PCR later on. Plant samples are a bit harder to analyze and we would have had to purchase various primers depending on the suspected species. This is due to the fact that plants have very little variation in their mitochondrial genome between species. The mitochondrial DNA is what we use in our experiment, and getting any data at all about what species some specific DNA came from hinges on the variation between species. In order to analyze plant samples, you look at the DNA in the chloroplast, not the mitochondria. Another reason for using animals, not plants, is that there are fewer introns scattered throughout the mitochondrial DNA of animals compared to plants, making amplification a more straightforward process.
In all we ended up with eleven samples suspected to contain animal DNA. Our prediction upon finding the samples as to the species of each is as follows-
1). Pileated Woodpecker
2). Dog (pet)
6). Single Amphibian Egg (Frog or Salamander)
8). Multiple Amphibian Eggs
Back in the lab it was time to begin our first step towards sequencing the DNA samples. Step one was to extract the DNA from the rest of the cellular material- RNA, Proteins, etc. We began with the scat samples because we used a separate procedure than with the non-scat samples. We used a procedure from an article entitled A Rapid and Simplified Procedure for Isolating DNA from Scat Samples, the authors being Shankaranarayana & Singh. The basic steps are as follows-
Scat DNA Extraction Protocol
- Add scat into a buffer.
- Three chemicals are added: SDS (sodium dodecyl sulfate), PVPP, and Chelex (to remove heavy metals and other molecules that might contaminate the DNA once we split the double helix structure and made it single stranded).
- Boil for 20 minutes.
- Extract DNA using mini DNA binding columns.
- Ready for PCR.
The protocol for out non-scat samples (insects, amphibian eggs, etc) was different from the scat samples. We began my grinding the samples in a glass-on-glass tube. After adding a portion of this to a buffer we used the following general steps to extract the DNA-
Non-Scat DNA Extraction Protocol
- Centrifuge in order to concentrate the biological matter in the bottom of the tube.
- Save resulting concentrated biological matter (includes everything in the cell; proteins, membranes, organelles, DNA, RNA, etc.
- Addition of Chelex.
- Float in 56°C water bath for 10 minutes.
- Float in 100°C water bath for 10 minutes
- Ready for PCR
We now had extracted DNA (both nuclear and mitochondrial) from both out scat and non-scat samples that were ready to amplify via PCR. PCR is the process of copying DNA with an enzyme called DNA Polymerase- hence “Polymerase Chain Reaction (PCR). The Polymerase only copies a section of the genome that we specify though, but using primers that tell the enzyme where to start and stop copying. Our primers tell the polymerase to copy a section of the mitochondrial genome called “Cytochrome Oxidase 1”. These were purchased and based on A Universal DNA Mini-Barcode for Biodiversity Analysis. Into our PCR reaction we added the following-
- A Primer Mix that we purchased from a company called Integrated DNA Technologies (IDT).
The primers were as follows-
- Uni-MinibarR1: 5'-GAAAATCATAATGAAGGCATGAGC-3'
- Uni-MinibarF1: 5'-TCCACTAATCACAARGATATTGGTAC-3'
- Extracted DNA.
- 1 Ready-To-Go PCR bead from GEHealthcare, which includes-
- Taq Polymerase
- Nucleotides (as the material for the amplification)
Our PCR cycle protocol was as follows-
95°C for 2 min
5 cycles of
95°C for 1 min
46°C for 1 min
72°C for 30 sec
35 cycles of
95°C for 1 min
53°C for 1 min
72°C for 30 sec
Final extension at 72°C for 5 min.
Running the PCR Product:
Once the PCR was complete we wanted to “run” the product (billions of copies of the cytochrome oxidase 1 gene) on an agarose gel using electrophoresis to test how successful the PCR was. Electrophoresis is the process of essentially pulling the DNA product across a resistant gel causing DNA fragments (hopefully the gene we amplified) of similar size to congregate and form dark bands that we can then analyze. For an image and explanation of our finished gels see “Results”.
Sequencing with VGN:
In order to sequence nucleotides in our amplified cytochrome oxidase 1 gene, we had to send a sample to the Vermont Genetics Network, a group that we have been collaborating with throughout this semester. At VGN, Tim Hunter sequenced our samples using a chain-terminating process. One of his coworkers, Heather Driscoll, drove the data to us as well as discussed our results. To analyze our data we plugged our sequence data into an online database at the NCBI website using a tool called BLAST.
The image below is the agarose gel that we ran the samples on. To the left of the gel (the one with no number) is the test row. This row is to show the ladder and how many base pairs are in each rung of the sample size. Based on these ladders, we can determine whether the enzyme worked in cutting the DNA at the correct sequence. Through this process, we can see that our extraction was successful and the remaining DNA samples can be sent to VGN.
(Insert gel image)
When Tim Hunter sequenced our samples, he was able to show the sequence and all the nitrogenous bases (As, Ts, Cs, and Gs) of the animal in question. When the sequence was brought to the class by Heather Driscoll, she showed us how to enter the information into the Basic Local Alignment Search Tool (BLAST). She enter one of the sequences into the database and through the magic of genetics and technology, within seconds we were able to determine what species the sequence came from.
From the ten samples given to VGN, the samples 1, 5, 6, 8, 10, and 11 (seven was not included because there was no DNA extracted) held the most DNA and could be sequenced. These results read as follows:
5: There were too many unknown sequences to get an accurate reading
8: Wolf Spider
10: Canine; either a dog (pet) or a Coyote
11: Canine; either a dog (pet) or Coyote
Throughout this class period the students were riveted and eager to learn more. We couldn’t believe that, what we thought was an amphibian egg, turned out to actually be a moth or a butterfly. At the end of this class period we knew how to start and end our own simplified version of the Barcode of Life project.
The DNA samples that were amplified enough to be sequenced were 1, 5, 6, 8, 10, and 11. Using BLAST, this tool identified sample 1 as a spider or a fungus. Although we predicted that the sequence belonged to a woodpecker, the scat displayed insect parts. There may have been more insect DNA than the possible woodpecker's DNA, so the insect DNA would have been amplifed more during the PCR process. The sequencing would have been from an insect and the BLAST tool identifying a different species then what we anticipated. Sample 5 has too many N’s in the sequence to get an accurate answer. An 'N' represents a particular location on the sequence that could be one base or another. For example, if there appeared to be an 'N' on a sequence of AGNU, the N could represent an A or C, depending on the species. The large quantities of N made identifying specimens and species unclear and difficult. Sample 6, according to BLAST claims it is most likely a butterfly or a moth. We assumed sample 6 belonged to an amphibian egg because it was found in a vernal pool. The primers that were used on all the samples do not work as well on amphibians, however, there are still primers being developed to accuratly target amphibian species. Sample 8 is a wolf spider, which was our most accurate prediction since we knew that the DNA we extracted belonged to a spider, as the physical spider was collected at Hogback. Lastly samples 10 and 11 belonged to a dog or a wolf, however, it is related to coyote.
By extracting DNA from an unknown species, we are able to narrow down what speciment belongs to what species. Although barcoding in the marketing world has a specific identification markers for all products, within genetics, scientist are analyzing DNA sequences of various species but have not discovered specific barcodes to identify organisms to the exact species. The barcode of life is a step towards categorizing all the various life forms on earth, looking at the uniqueness of all organisms on earth on a molecular level.
-Meusnier, Singer, Landry, Hickey, Hebert, Hajibabaei. 2008. A Universal DNA Mini-Barcode for Biodiversity Analysis. BMC Genomics. http://www.biomedcentral.com/1471-2164/9/214
-Shankaranarayana. Singh. 1988. A Rapid and Simplified Procedure for Isolating DNA from Scat Samples. Current Science. Vol 75: No9.
-Vermont Genetics Network: Tim Hunter. Heather Driscoll.