DNA preservation on blotting paper & DNA extraction protocol for field collection of coral samples suitable for marker gene sequencing approaches

DNA preservation on blotting paper & DNA extraction protocol for field collection of coral samples suitable for marker gene sequencing approaches Marlen Schlotheuber 1, Kok Lynn Chew 2, Sebastian Szereday 2, Christian R. Voolstra 1 Affiliations 1 Department of Biology, University of Konstanz, Konstanz, Germany 2 Coralku Solutions, Non-Profit Organization for Coral Reef Research and Restoration, Kuala Lumpur, Malaysia. Summary Collecting biological samples from remote field settings presents logistical challenges in preserving and transporting DNA to laboratory facilities for further processing (e.g., DNA extraction, PCR amplification, and sequencing). To address this, we tested preserving DNA samples on blotting paper, offering a practical and efficient solution for transporting biological samples as a dry medium. Blotting papers, commonly used for the transfer and immobilization of nucleic acids and proteins, have high binding affinity and absorption capacity, making them suitable for DNA preservation. Coral samples, in the form of sprayed off tissue in DESS buffer [1], are pipetted onto blotting paper for storage and transport. Using the Qiagen Dneasy Plant Pro Kit that entails a bead-beating step to effectively lyse the samples, we obtained sufficient DNA for subsequent marker gene sequencing approaches. This method enhances the feasibility of conducting genetic research in remote locations by overcoming the limitations associated with liquid sample preservation and transportation. Protocol (step-by-step): Sample storage in DESS buffer on blotting paper - collect coral fragments (~2-4 cm2 surface) - incubate in DESS buffer and spray off tissue following previous protocol [1] - Mix tissue slurry using a mini vortex mixer (4,000 rpm) for 1 minute or until the slurry color is homogenous with little pellet remaining - Pipette 200 µL of the tissue slurry onto blotting paper (we use: Gel-Blotting-Papier of size 150 x 200 mm, thickness 0.8 mm, e.g. link; Note: We recommend 200 µL for a medium sized fragment (~ 2-4 cm2 surface) and 500µL for a small sized fragment (< 2cm2 surface) to ensure sufficient DNA. Note: Label blotting paper with sample names prior to transferral; for efficient use of space, we split blotting paper into grids of ~4x4 cm by drawing lines using a pencil and ruler (FIgure 1). - Place blotting paper on a clean surface and let it dry at room temperature. Note: Avoid direct sunlight exposure. - Place the dried blotting paper into a ziplock plastic bag to reduce the risk of contamination. Note: The plastic bag with the dried blotting paper should be kept at 4°C for longer-term storage to reduce the risk of DNA degradation. For short-term storage, the samples can be kept at room temperature (travelling time). Add some silica beads to avoid moisture build-up and make sure the plastic bag is properly sealed. Figure 1. Transferred and dried tissue slurry on the blotting paper. DNA extraction using the DNeasy Plant Pro Kit 1. Cut out the dried tissue slurry area of blotting paper 2. Cut the respective sample tissue area into small pieces using a sterile scalpel and transfer into the disruption tube. Note: wash scalpel in between samples using 20% bleach, VE water, 70% ethanol, VE water to avoid/minimize cross-contamination 3. Add 600 μl Solution CD1 to the 2 ml tissue disruption tube. Vortex briefly to mix. 4. Homogenize using the PowerLyzer24Homogenizer (Setting: S: 1700, C:1, T:1min) 5. Centrifuge the tissue disruption tubes at 12,000 x g for 2 min. 6. Transfer the supernatant to a 1.5 ml collection tube. Note: Expect 350–450 μl. Quickly pipette the supernatant out of the collection tube since the paper starts to soak up the liquid. When processing many samples, we recommend to centrifuge the samples in smaller batches. 7. Add 200 μl Solution CD2 and vortex for 5 s. 8. Centrifuge at 12,000 x g for 1 min at room temperature. Avoiding the pellet, transfer the supernatant into a new 1.5 ml collection tube. 9. Add 500 μl Buffer APP and vortex for 5 s. 10. Load 600 μl of the lysate onto an MB spin column and centrifuge at 12,000 x g for 1 min. 11. Discard the flow-through and repeat step 9 to ensure that all lysate has passed through the MB spin column. 12. Carefully place the MB spin column into a clean 2 ml collection tube. Avoid splashing any flow-through onto the MB spin column. 13. Add 650 μl Buffer AW1 to the MB spin column. Centrifuge at 12,000 x g for 1 min. 14. Discard the flow-through and place the MB spin column back into the same 2 ml collection tube. 15. Add 650 μl Buffer AW2 to the MB spin column. Centrifuge at 12,000 x g for 1 min. 16. Discard the flow-through and place the MB spin column into the same 2 ml collection tube. 17. Centrifuge at up to 16,000 x g for 2 min. Place the MB spin column into a new 1.5 ml collection tube (provided). 18. Add 50µl (50–100 μl) of Buffer EB to the center of the white filter membrane. 19. Centrifuge at 12,000 x g for 1 min. Discard the MB spin column. The DNA is now ready for downstream applications. 20. Assess DNA quantity and quality and store DNA at -20°C. Expected yield is 2-90 ng/µL. References 1. Voolstra CR, Colin L, Dörr MS, Perna G, Fiesinger A, Cárdenas A. DNA preservation & DNA extraction protocol for field collection of coral samples suitable for host-, marker gene-, and metagenomics-based sequencing approaches. Zenodo; 2025. doi:10.5281/ZENODO.6523532 Acknowledgements MS and CRV are supported by the German Research Foundation (DFG), project: 468583787. KLC, SS, CRV are supported by G20 Coral Research & Development Accelerator Platform (CORDAP), Coral Accelerator Program 2022 (CAP), Project ASSIST, grant: CAP-2022-1591.