Modified DNA Extraction Protocol for Identifying Ciliate Biodiversity By Evelynne Morris, Catherine Pivalizza and Kirubel Tigabu Department of Biology, Baylor University, Waco, TX 76798 INTRODUCTION METHODS AND MATERIALS DISCUSSION The results from the gel image displayed bands on the PCR product of the eDNA sample and the positive control sample which were approximately 450+ bp in size (Table 1). The V4 primers used specifically allowed us to examine the band on the gel which was amplified by the primers in the 18SV4 region where the band was located and picked up on the gel imaging device (Figure 1). In the PCR step of our protocol, two different protocols were used. The researchers who used the Chelex protocol had a better chance of getting positive DNA results in the samples. Eventually, these changes to the protocol allowed there to yield sufficient results and attain DNA from our soil samples. There were some limitations in our approach that should be discussed and improved upon. One weakness that was very difficult to avoid entirely was human error. This source of error can be seen when pipetting the control samples and the environmental DNA into the wells of the gel. This can be avoided by having the same researcher pipette all the samples in the wells in order to not have a discrepancy in the results. A major source that can attributed to setting up the PCR reaction. Studying these soil ciliates and finding protocols to help extract their DNA can lead to the identification to new soil organisms hence detect biodiversity. From here, the next step in this protocol would be to do Illumin based NextGen sequencing in order to sequence the V4 region of the sample. The sample will then go through metabarcoding and see the biodiversity of eDNA in our soil sample. Acting as predators of bacteria and protozoa, ciliates play a key role in the ecosystem as phagotrophic protists. [2, 6] Through their diversity, each species contribute to their present environment in an alternate way. Due to few distinguishments in their features, most of the biodiversity remains unknown. With advancing technology, such as polymerase chain reaction (PCR) and gel electrophoresis, research in this field continues to increase but genome databases are still incomplete. [6] Through Ludox Centrifugation, DNA extraction, PCR amplification, and gel electrophoresis, the sampled ciliate genes can undergo metabarcoding. [1, 4] From the specific DNA provided for PCR, the amplified segments become visual in the agarose gel. [4] Specifically, the Variable Region 4 (V4) ribosomal ciliate DNA assist in this amplification process. [3] In this study, 18SV4 amplifies the Signature Region 2 (SR2) and Cox1 as primers. By extracting the DNA from the soil and amplifying the DNA with PCR primers, taxonomic identification can proceed. This sequencing of environmental samples uses short DNA barcodes to create an inventory of all given life forms. [5] Because gene copies vary greatly among taxa, collecting morphological metadata makes it easier for scientists to identify key evolution mutations in the DNA. [1, 5] To explain why ciliates are key biological protists, the genetics behind them needs to be understood. For this report, we will compare the collected samples through PCR and gel electrophoresis ran by modified DNA extraction protocol. By metabarcoding ciliates, scientists can infer why certain species differed from others. Adding to the genome database will contribute to learning the science behind ciliates and how they aid in the environment. -Ludox HS40 -2 mL soil and D.I. water -Centrifuge for 15 min. at 4300 xg -Cell count (organismal) -10 g filtered soil -Add 368 µL Glutaraldehyde -OB Protease Solution -BL Buffer (Guanidine Hydrochloride) -100% Ethanol (Binding: DNA Column) -HBC and Wash Buffers -Elution Buffer -0.3 g soil -Solutions C1, C2, C3, C4, C5, and C6 -Vortex and Centrifuge at 10,000 xg -Incubate at 4°C -5% Chelex -Proteinase K -Incubate 30 min in 56°C heat block, 8 min in 100°C water bath -Centrifuge 3 min at 16,000 xg -1.8% Agarose and 1X TAE Buffer -2 µL EtBr -Set for 25 min -Run for 30 min at 100V -DNA Concentration (ng/µL) -Purity Ratios -A260/A280 -A260/A230 RESULTS REFERENCES Bik, H., Porazinska, D., Creer, S., Caporaso, J., Knight, R., & Thomas, W. (2012, January 11). Sequencing our way towards understanding global eukaryotic biodiversity. doi: 10.1016/j.tree.2011.11.010 Dopheide, A., Lear, G., Stott, R., & Lewis, G. (2009, August 01). Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods. doi: 10.1128/AEM.00412-09 Chantangsi, C., Lynn, D. H., Brandl, M. T., Cole, J. C., Hetrick, N., & Ikonomi, P. (2007). Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena. Int. J. Syst. Evol. Microbiol., 57(Pt 10), 2412–2425. doi: 10.1099/ijs.0.64865-0 Garibyan, L., & Avashia, N. (2013, March). Research Techniques Made Simple: Polymerase Chain Reaction (PCR). doi: 10.1038/jid.2013.1 Morard, R., Garet-Delmas, M., Mahé, F., Romac, S., Poulain, J., Kucera, M., & Vargas, C. (2018, February 07). Surface ocean metabarcoding confirms limited diversity in planktonic foraminifera but reveals unknown hyper-abundant lineages. doi: 10.1038/s41598-018-20833-z Murase, J. (2017). Quest of Soil Protists in a New Era. Microbes and Environments Microbes and environments, 32(2), 99-102. doi: 10.1264/jsme2.ME3202rh Figure 1 - The gel image of the DNA from the BioRad Imager ACKNOWLEDGEMENTS The initial Ludox Centrifugation procedure resulted in no cells, so Glutaraldehyde was added in the modified procedure. EZNA DNA Extraction produced no beneficial results. Half of the class used the MO-BIO PowerSoil DNA Extraction Kit while the other half used the Chelex DNA Extraction Kit in the first trial The Chelex DNA Extraction Kit was used in the final protocol, producing results in Gel Electrophoresis and Nanodrop pictured to the left. Dr. Tamarah Adair, Senior Lecturer Ankan Chouhury, Lab Assistant Will Mullen, Lab Assistant Nanodrop Results (ng/µL) 260/280 260/230 Amount (ng) of DNA added to the reaction PCR - PCR + PCR eDNA Size of + eDNA band 94.9 1.03 0.28 no band band 450 plus larger Scan the QR Code to view detailed entries from our virtual class notebooks Table 1 - DNA Concentration and Purity Ratios