Indeterminate PCR Test: visiting e-DNA lab for Kanagawa Prefecture 2

 

From a kind of easy guess of an amateur, it seems to me comprehensive detection of e-DNA is more complicated than the species-specific detection. If the thing somebody likes to know is the existence of a particular species in a pond, and the DNA sequence of the animal is known, we can just search for the chain clearly indicating the owner of this DNA arrangement. For a comprehensive search, the story would be different. For one thing, there is no “comprehensive” database for DNA whose owner can leave a bit of theirs in river, pond, lake, sea, … Such database is owned by the God, don’t you think? So, what the researchers can do, at least with today’s knowledge at hand, is finding a series that would represent the characteristics of a family, especially when the family have numerous members. The scientists do like this: as for the species-specific detection, researchers for comprehensive detection add the probe of fluorescence to identify the existence of a family within the sample. If there is a particular color or a pattern of emission of light, they can think the family of these creatures exists within the water. But how to read the strength of color or light from the PCR? Lots of illumination signify … lots of bugs of that family for sure, but lots of one particular species? or the variety within the family? or else? That’s a huge academic topic. This is the difficulty at the exit stage of analysis. But actually, the complication starts before that, from the beginning.

PCR machines in the Centre

For one thing, the time required for PCR to comprehensive detection is undeniably longer. Mr, Hasebe said “As the process of PCR is definitely more cumbersome than the species-specific case, I need at least 4 days to arrive at the analysis stage of the study for a sample.” Not only that, there still are species that do not have a proper primer manufactured for augmenting the target DNA sequence, like Ayu fish and Lampetrinae. If there is no suitable primer, PCR will not detect the existence of them, right? Then, there comes a problem of non-specificity in DNA sequence for a family. A family of taxonomy having lots of species in its umbrella is not like a clear-cut manifestation of features for a species. I guess it’s like “You love Mrs. Green Apple, and I’m crazy for SixTones (er, they have #1 and 2 positions for YouTube trending in J-pop category as of January 21). Now, in terms of DNA sequence, they both have J-pop DNA qualities that would be shown in the distributional pattern of nucleobase.” I.e. We would say Mrs. Green Apple can be in J-pop category in such-a-such probability, and so should be for SixTones. That’s far from a clear-cut conclusion of mathematical question with QED.

The result of one PCR ready for close inspection.

So, Mr. Hasebe regularly sits in front of his PC for long hours to check the PCR analysis showing a statistical curve for one DNA array matching the baseline-distributional pattern for one family. If the result does not fit well to the criterion curve, he returns to PCR to multiply more the target nucleobase hoping the next analysis produces more fit to the reference. “That’s where researchers are required to decide if a creature of a family exists in the sample, and in the end the environment where the sample comes from,” he said. I asked him if it is possible to automatize the decision which would make the job of the researcher easier. He answered, “Nay, at least in today’s technology. In the end the final part of examination is still done by human brain. Besides, the DNA database for creatures even at the family level is hopelessly incomplete. When the reference point is at the developmental stage, researchers must do with that constraint, you see?” Hmmmmmm …

The target sequences come out in such a distributional way.
Now, it’s your turn, human brain!

Until I became a forest instructor, I had a sort of romantic admiration for natural science. A disclosure of personal info: I’m a social scientist. We were hearing a quiet murmur in our heads what we’re measuring could be a complete illusion of society made of very human intentions. Can the numbers obtained from such things be objective, natural, reliable data worth analyzing by mathematical equations as science? But Mr. Hasebe grumbled to us it was sometimes laborious work to “purify” DNA data to identify the base sequence. After such an operation they conclude and catalogue the contents of biodiversity in our prefecture. … Oh. Then, it’s not much different from social scientists doing before checking the validity of a model, so I thought. It’s fun experiencing such wonders, endless wonders appearing from under our forest.

Next week, I tell you my understanding for the position of Kanagawa Prefecture’s effort to cataloguing biodiversity, including e-DNA approach. Please stay tuned 😊

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 

https://www.pref.kanagawa.jp/docs/b4f/index.html