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New Techniques for eDNA Detection

Yesterday and today I have been lucky enough to be offered a chance to work with Carina Davis, a molecular technician in the GEM Lab (an amalgamation of the Gamete, Ecotoxicology & Molecular Laboratories). Carina specialises in “new technologies”, and she often works with detecting environmental DNA (eDNA) – the DNA, or fragments of, that are left behind by organisms in the environment. This can be a useful tool to determine the presence of certain organisms, when you are unable to view or obtain the organism yourself. For example, eDNA can be found in shed skin, gametes, faeces, and mucus. The extraction of eDNA is well-documented in permafrost, freshwater, and seawater. More information can be read in this article:

eDNA

A quick Web of Science search for the topic keyword “eDNA” in journals (post-2010) found 532 results. Of the first 40 results, only two of them were not investigating eDNA found in water. One sampled residual bear saliva left on partially consumed salmon, and the other sampled soil in pastoral agriculture looking for evidence of biological processes. None were about eluting eDNA from dry terrestrial surfaces.

manuka-white-flower-upcloseCarina was contacted by a researcher who wanted to find out if having a bee hive close to mānuka affected the types of pollinators that visited the flowers. I guess the researcher could have sat next to a mānuka tree and visually record every single bug that visited, but perhaps an easier way would be to look for eDNA from the insects that visited the flowers. This was a whole new method of determining pollinators, so Carina looked for  any similar research carried out to detect eDNA to help her come up with a method to trial. She found one journal article which used eDNA to investigate the microbial community present on flowers, before and after pollination (DNA from the bacteria that live on insects):

eDNA of pollinators

GEM lab me2

Using the techniques in this article as a guide, we removed surface material (and hopefully some DNA) from a few samples of mānuka flowers (two from an area with a hive, and two without; one of each stored frozen, and the other in ethanol). After a lot of steps adding solutions, filtering through a tiny 0.45μm membrane, vortexing, and centrifuging, we finally had four small vials of colourless solution. This took almost a day to achieve, and each vial only contained around 20μL! What really surprised me was the tiny volumes these scientists are working with. This did mean that I got to use all their fancy pipettes and look vaguely science-y. After storing in the fridge overnight, we prepared our samples for DNA amplification. This involved adding a few more chemicals (such as primers which will later bind to sections of DNA that we are trying to find), then removing only 2μL of each to place into a PCR machine. The machine causes any DNA in the sample to be multiply by forcing it to repeat its replication cycle many times over. The PCR process took just over an hour. After this, we prepared the samples for electrophoresis and made up a gel plate.

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Carina’s colleague using the UV lightbox after electrophoresis of her buttercup DNA samples

Unfortunately, very little DNA (nucleic acids) appeared to be found once we placed the completed gel into a UV light box. We then carried out a quick test to quantify how much DNA was present (by absorbance/optical density), and all but one sample was “too low to determine”. We had a discussion and reviewed the methods in the paper by Ushio et al. They appeared to use a whole florescence rather than small individual flowers as we did. This could be a possible reason for our lack of DNA when compared to their results. Carina will do some further investigation after the weekend.

UPDATE: In our first trial, Carina used a soils kit and  the associated primers that would indicate the presence of microbial DNA. She repeated the procedure a few days later, but this time using primers designed to indicate invertebrate DNA. Although we knew our sample was very minute, and therefore very difficult to remove and detect DNA, she was somewhat successful in showing that invertebrates had left eDNA behind on one or more specimen. The image below, produced under UV light, shows eight wells at the top of the gel (agarose) plate, the two outside wells contain a DNA ladder for reference. Wells 2 to 5 contained our sample extractions, Well 6 was a control solution made from filtering distilled water through the same membrane, then applying all the same techniques as the real samples. Finally, Well 7 was a blank that was also put through the PCR process. You can see that this sample showed no DNA presence, as expected. Some DNA was present in the  others, although not expected in the Well 6 sample. The much darker bands shown coming from Wells 3&4 suggest that invertebrate eDNA was detected in these samples, particularly from Well 4.  The sample in Well 4 was taken from a mānuka flower in an area that contained bee hives (so, it’s not exactly surprising that it had been visited by invertebrates!), kept frozen and then had a phosphate buffer added. The next stage for Carina will be to determine the species that visited this flower.
GEM lab DNA image numbered (1).jpg

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