TITLE: QIAamp DNA Extraction Protocol
AUTHORS: Vicky Ooi, Lee McMichael, Margaret E. Hunter, Aristide Takoukam Kamla, Janet M. Lanyon

[DESCRIPTION]
Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new ‘High Volume­­- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol’ (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of SNP markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.

[GUIDELINES]
Follow the guidelines as per the "Protocol: Isolation of DNA from Stool for Human
DNA Analysis" from the QIAamp Fast DNA Stool Mini Handbook (Version: February 2020) unless noted otherwise.

[STEPS]
SECTION: Faecal Sampling and Processing
1. Use a sterile blade to scrape off 220 mg of faecal material from the outside surface of a stool and then transfer it into a 2 mL microcentrifuge tube.

220 mg

SECTION: Faecal Sampling and Processing
2. Transfer the faecal material into a mortar and grind the faeces into powder with liquid nitrogen.
SECTION: Cell Lysis, Protein Digestion, and Purification
3. Add 500 μL of InhibitEX buffer to the mortar containing the faecal material to further grind and mix in the buffer with the ground faeces. Transfer the liquid back into the 2 mL tube.

500 µL

SECTION: Cell Lysis, Protein Digestion, and Purification
4. Add another 500 μL of InhibitEX buffer to the mortar to mix in any leftover faecal material on the mortar. Transfer the liquid back into the 2 mL tube.

500 µL

SECTION: Cell Lysis, Protein Digestion, and Purification
5. Vortex continuously for 1 min or until the solid material is thoroughly homogenised.

1 min

SECTION: Cell Lysis, Protein Digestion, and Purification
6. Centrifuge the sample at 20,000 g (~14,000 rpm) for 2 min to pellet stool particle.

2 min

SECTION: Cell Lysis, Protein Digestion, and Purification
7. Pipette 25 μL of Proteinase K into a new 2 mL tube.

25 µL

SECTION: Cell Lysis, Protein Digestion, and Purification
8. Pipette 800 μL of supernatant from the centrifuged homogenate into the 2 mL microcentrifuge tube containing Proteinase K.

800 µL

SECTION: Cell Lysis, Protein Digestion, and Purification
9. Add 800 μL of Buffer AL to the mixture and vortex for 15 s.

800 µL
15 s

SECTION: Cell Lysis, Protein Digestion, and Purification
10. Incubate at 70°C for 10 min. Then, centrifuge briefly to remove drops from the inside of the tube lid.

70 °C
10 min

SECTION: Cell Lysis, Protein Digestion, and Purification
11. Split the lysate into two 2 mL tubes (~813 μL each tube).
SECTION: Cell Lysis, Protein Digestion, and Purification
12. Add 400 µL of 99.9 % ethanol to both tubes containing the lysate (thus, 800 µL of ethanol overall) and mix by vortexing.

400 µL

SECTION: Cell Lysis, Protein Digestion, and Purification
13. Carefully apply 600 μL of lysate to the QIAamp spin column. Close the cap and centrifuge at 20,000 g (~14,000 rpm) for 1 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the tube containing the filtrate.

600 µL
1 min

SECTION: Cell Lysis, Protein Digestion, and Purification
14. Repeat step 13 until all the lysate has been loaded on the column.
SECTION: Cell Lysis, Protein Digestion, and Purification
15. Carefully open the QIAamp spin column and add 500 µL of Buffer AW1. Centrifuge at 20,000 g (~14,000 rpm) for 1 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the collection tube containing the filtrate.

500 µL
1 min

SECTION: Cell Lysis, Protein Digestion, and Purification
16. Carefully open the QIAamp spin column and add 500 µL of Buffer AW2. Centrifuge at 20,000 g (~14,000 rpm) for 3 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the collection tube containing the filtrate.

500 µL
3 min

SECTION: Cell Lysis, Protein Digestion, and Purification
17. Centrifuge at 20,000 g (~14,000 rpm) for 3 min to eliminate the chance of possible Buffer AW2 carryover.

3 min

SECTION: Elution
18. Transfer the QIAamp spin column into a new, labelled 1.5 mL microcentrifuge tube and pipette 100 µL of Buffer ATE directly onto the QIAamp membrane to elute the DNA from the spin column into the 1.5 mL Eppendorf LoBind microcentrifuge tube. Incubate for 1 min at room temperature, then centrifuge at 20,000 g (~14,000 rpm) for 1 min to elute DNA.

100 µL
1 min: 15 °C to 25 °C
1 min

SECTION: Storage of DNA extracts
19. Store the DNA isolate at -20°C for use within a week or at -80°C for longer-time storage.

-20 °C
-80 °C