Extraction of Diatom DNA from Water Samples and Tissues

Our research mainly focuses on the topic of drowning. We wanted to answer the question of whether the body found in the water died from drowning, as well to answer another question of where person fell into the water. That means to know the location of the drowning.

Compared with other technologies, we provide a simple, convenient, and economical diatom DNA extraction protocol that we can use better in forensic research, especially in basic forensic practice. Our laboratory will further focus on the drowning-related problems based on the pattern in the water, such as reason of death, post mortem interval, and so on. What's more, we also carry on the research through the forensic entomology and some special phenomenons in the tropical forensic medicines.

To begin, take 10 milliliters of the diatom water sample in a centrifuge tube. Centrifuge the tube at 13, 400 g for five minutes. With a pipette gun, carefully discard 9.8 milliliters of the supernatant.

Then transfer 200 microliters of the enriched diatom water sample into a two milliliter centrifuge tube. Next, take 0.5 grams of lung margin tissue from a drowned human body. With scissors, cut the lung tissue until it becomes muddy.

For DNA extraction off both diatom and lung samples, first add glass beads to both samples. After mixing the samples well, add 40 microliters of Proteinase K to the tubes. After a 15-minute incubation at room temperature, add 200 microliters of binding buffer to both tubes.

Immediately mix the samples with a vortex mixer for four minutes. Then place the tubes in a water bath at 70 degrees Celsius for 10 minutes until the solution becomes clear. Next, transfer the clear solution with flocculent precipitate into a three-centimeter-long absorption column packed with silicon matrix.

Centrifuge the column at 8, 000 g for 30 seconds. Then discard the waste liquid in the collection tube, and put the column back into the collection tube. Now, add 500 microliters of the inhibitor removal buffer into the column, and centrifuge at 13, 400 g for 30 seconds.

After discarding the waste liquid, add 700 microliters of the washing buffer to the column, and centrifuge again. After discarding the waste liquid, place the absorption column back into the empty collection tube. Centrifuge the column at 14, 500 g for two minutes to remove as much wash buffer as possible.

Now, take the column out and place it in a clean centrifuge tube. Add 100 microliters of elution buffer to the middle part of the absorption membrane. Centrifuge the column at 13, 400 g for one minute after a five-minute incubation at room temperature.

Transfer the solution obtained in the collection tube back into the absorption column before centrifuging again. Store the eluted diatom DNA at 2 to 8 degrees Celsius for future use. Agarose gel electrophoresis showed diatom DNA bands between 250 to 500 bp in both water samples and tissues after PCR amplification.