5:15 PM - 6:45 PM
[ACC27-P07] Investigation of sample enrichment for the analysis of ancient fungal DNA in Antarctic ice cores
Keywords:microorganism, ancient DNA, population genetics, evolution
It is important to clarify variations in the number of organisms and species composition due to past global environmental changes in order to obtain basic information to consider effects of global warming biodiversity and responses to biological conservation. Information about past organisms can be obtained from fossils preserved in sediments. However, genetic information cannot be obtained because DNA, which is organic material, has been degraded. Microorganisms such as fungi and bacteria that were transported to Antarctica in the past have been frozen in the ice sheet over the past several hundred thousand years.
The final goal of this study is to develop a method to extract ancient fungal cells from ice cores drilled at the Dome Fuji Station and analyse these DNA sequences. However, since the concentration of cells in the sample is low, methods that are reliable, rapid, and effortless to collect the fungal cells are required. This study investigated an method for extracting the cells from samples using a dielectrophoresis-based instrument (ELESTA, AFI Corp., Japan). Since the sample introduction volume of this device is limited to a few milliliters, the sample must be concentrated beforehand. In addition, for DNA analysis, the final volume must be reduced to a few microliters. This study used snow collected from a glacier in Siberia, Russia.
The fungal cells were collected by placing a 25-mL melt water sample in a 50-mL centrifuge tube, centrifuging (8,000 g, 20 min), and discarding 20 mL of the supernatant solution. Then, 5 mL of the remaining sample was introduced into ELESTA. The cells were collected in the microfluidic channels of the ELESTA chip in the form of a glass slide and could be observed under a microscope. If fungal cells were found, the cells were collected from the ELESTA chip along with 140µL of water and transferred to a 0.2µL PCR tube. The sample solution was then concentrated to 5 µL on a dry block heater. The above operation enabled easy concentration of the sample for DNA analysis. However, the microscopic observation was sometimes difficult to distinguish between the cells and impurities. We think the cells should be stained prior to introduction into ELESTA.
The final goal of this study is to develop a method to extract ancient fungal cells from ice cores drilled at the Dome Fuji Station and analyse these DNA sequences. However, since the concentration of cells in the sample is low, methods that are reliable, rapid, and effortless to collect the fungal cells are required. This study investigated an method for extracting the cells from samples using a dielectrophoresis-based instrument (ELESTA, AFI Corp., Japan). Since the sample introduction volume of this device is limited to a few milliliters, the sample must be concentrated beforehand. In addition, for DNA analysis, the final volume must be reduced to a few microliters. This study used snow collected from a glacier in Siberia, Russia.
The fungal cells were collected by placing a 25-mL melt water sample in a 50-mL centrifuge tube, centrifuging (8,000 g, 20 min), and discarding 20 mL of the supernatant solution. Then, 5 mL of the remaining sample was introduced into ELESTA. The cells were collected in the microfluidic channels of the ELESTA chip in the form of a glass slide and could be observed under a microscope. If fungal cells were found, the cells were collected from the ELESTA chip along with 140µL of water and transferred to a 0.2µL PCR tube. The sample solution was then concentrated to 5 µL on a dry block heater. The above operation enabled easy concentration of the sample for DNA analysis. However, the microscopic observation was sometimes difficult to distinguish between the cells and impurities. We think the cells should be stained prior to introduction into ELESTA.