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One of
the labs for e-DNA analysis at Environmental Research Center |
According to Mr. Yuta Hasebe, the Chief Researcher for Water Source Environment Analysis, these days there are private businesses that contract the species-specific detection method. That is to verify if a certain species exists in one particular aquatic environment for how much of their volume. Actually, I know the City of Yokohama hired such consultant some 2 years ago to check the situation of Montane brown frog in Niiharu Citizen Forest 新治市民の森. Mr. Hasebe added in 2023 kids from a couple of high schools in the Prefecture knocked on the door of Environmental Research Center to learn and do species-specific detection for their target species. They published the result of the research during the national competitions of scientific study by the highschoolers. They won the prizes. The level of e-DNA has reached this level in Kanagawa Prefecture. Wow. That’s said, the comprehensive detection method for assessing biodiversity of an environment is still at the pre-business stage, Mr. Hasebe said. The reason? Please keep on reading.
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| A
summary of the study done by Kamimizo-Minami High 県立上溝南高校 which reported at the national conference of Japanese Society for Preservation of Birds. The kids did species-specific detection for Lefua echigonia, a fish listed as EN for Japanese red list by the Ministry of Environment. |
During the tour, first we went in the lab to filter DNA from the sample we brought in. There, the samples are filtered by motored strainers, then warmed at 56°C to be concentrated and purified for DNA using centrifuge and chemicals. In the process the DNA is harvested in test tubes. Mr. Hasebe said “The theory these machines use is the same as you do in the field with syringes and cartridges. The difference is, aside from mechanized power of the sieve and the centrifuge, collected DNAs are treated in the environment that can prevent the collapse of genetic alphabet. The temperature centrifuge operates is at just right. As we work with the sample one-by-one, each purified test tube must wait for their turn before going to PCR. So, we use powerful deep freezers for them until we’re ready to process them. You see? COVID-19 forced the Prefectural Government purchasing lots of high-end freezers for vaccination. Now some of them have retired from medical emergency, and we can introduce them to our lab, FREE! (a big grin)” Well, it’s really the Unintended Consequence. COVID is contributing to our community cataloguing the biodiversity. Not bad …(er …?)
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| The water is being filtered here. |
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| The filtered water is “sanitized,” and |
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| Put in a deep freezer until DNA extraction process. |
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| It’s the freezer by courtesy of COVID-19. |
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| Centrifuge.
The Center has several of it. DNA is extracted from mitochondria. |
The next step is Polymerase Chain Reaction, aka now-very-familiar-word PCR. It is the method invented by Dr. Kary Mullis who won the Nobel Prize for it in 1993. … I’m not sure if I can summarize it correctly, but let me try … First, the above purified DNA is heated at 94°C to untangle its doble helix structure, then the temperature of the DNA rope (or noodle maybe) is lowered to 60°C. Next, a massive dose of primer and polymerase is added. Primer is a chain of nucleobase that can connect to the part of DNA which signals the owner of it is such-a-such species, or from a particular family. Finally, the sample is heated again to 72°C in order for targeted parts of a DNA chain to be multiplied with a help of polymerase through connecting to lots of poured primer. By repeating such procedure for n>20 times, the intended segment of DNA will be multiplied to 2n pieces. For Covid-testing, the aimed DNA segment was of the virus. For environmental DNA analysis, the part of DNA to be augmented is the marker of a particular creature, being it at the species level (species-detection) or for class, order, and/or family level (comprehensive detection). In the process the target DNA is increased to the level unseen in natural world. It can contaminate the not-yet PCR-ed sample, and the environmental analysis should be ruined. So, the manuals for e-DNA from the Ministry of Environment, and by e-DNA Society of Japan recommends the practitioners to separate labs between DNA extraction room and PCR place. After showing us the process of DNA extraction, Mr. Hasebe introduced us to the next-door room for PCR and actual analysis.
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| PCR machine next door |
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| The bottles waiting for the next field study … |
For the measures of e-DNA for environmental issues of Kanagawa Prefecture, please make a contact with Kanagawa Environmental Research Center 神奈川県環境科学センター
1-3-39 Shinomiya, Hiratsuka City, 254-0014
〒254-0014平塚市四之宮1-3-39
Phone: 0463-24-3311
FAX: 0463-24-3300
k-center@k-erc.pref.kanagawa.jp
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