Ancient DNA Sampling: Preventing Contamination

Ancient DNA (aDNA) research is extremely sensitive because the DNA from long-dead organisms is fragmented and present in tiny amounts. Even a single skin cell from a modern human can overwhelm the ancient signal, leading to false results. This guide covers the standard protocols researchers use to prevent contamination during sampling, from field collection to the laboratory extraction step. You’ll learn the key principles of sterile technique, dedicated clean-room practices, and contamination monitoring.

What You’ll Need

• Personal protective equipment (PPE): disposable full-body coveralls, shoe covers, hairnets, beard covers, face masks (N95 or higher), and two pairs of nitrile gloves.
• Clean-room or dedicated aDNA laboratory with UV irradiation capability and positive air pressure.
• Sterile, DNA-free consumables: sterile scalpel blades, forceps, drill bits, and sample tubes.
• DNA decontamination solutions: 10% bleach (sodium hypochlorite) and 70% ethanol.
• UV crosslinker or UV lamp for sterilizing surfaces and tools.
• Negative controls (blank samples) and positive controls (known ancient or modern DNA) for monitoring.
• Clean, dedicated tools for each sample to avoid cross-contamination.
• Gloves and masks that are changed frequently.

Step-by-Step

1. Plan and prepare before entering the clean area. Write down the sampling plan, label all tubes and containers, and prepare all reagents and tools. This minimizes the time and movement inside the clean room, reducing the chance of introducing contaminants.
2. Don full PPE before entering the clean lab. Put on coveralls, shoe covers, hairnets, beard covers, and a face mask. The mask must cover both nose and mouth to prevent respiratory droplets from reaching the sample. Wear two pairs of gloves; the outer pair is for handling the sample, the inner pair is a backup if the outer tears.
3. Decontaminate the work surface and all tools. Wipe down the workbench with 10% bleach, then 70% ethanol. Turn on the UV lamp for at least 15-30 minutes to crosslink any residual DNA on surfaces and tools. Allow surfaces to dry completely before starting.
4. Clean the exterior of the sample. If the sample is a bone, tooth, or sediment, remove the outer layer using a sterile scalpel or drill. This step removes surface contamination that may have accumulated during excavation or handling. For soil or sediment, take material from the interior of the block if possible.
5. UV-irradiate the cleaned sample. Place the sample in a UV crosslinker for 5-10 minutes on each side. UV light damages DNA, so this step reduces any remaining surface DNA. Be careful not to overexpose the sample, as UV can also damage the ancient DNA inside.
6. Sample the interior material. Using a new, sterile scalpel or drill bit, take a small piece from the interior of the bone or tooth. For teeth, the inner pulp or cementum is often preferred. For bones, the dense cortical bone is better than the spongy interior. For sediment, take a core from the center of the block.
7. Place the sample in a sterile tube. Immediately transfer the sampled material into a pre-labeled, sterile tube. Do not touch the sample with gloved hands; use sterile forceps or the scalpel blade. Close the tube securely.
8. Change gloves and tools between every sample. After each sample, discard the outer gloves, scalpel blade, and any other tools that touched the sample. Put on a new pair of outer gloves and use new sterile tools for the next sample.
9. Process negative controls alongside your samples. Open a blank tube and perform the same steps (e.g., wipe with a sterile swab) without adding any sample. This blank will be processed through the DNA extraction and sequencing pipeline to detect any contamination that occurs during those steps.
10. Document everything. Record the sample ID, date, time, who handled it, and any deviations from the protocol. This chain of custody is essential for interpreting results and troubleshooting if contamination is detected.
11. Store the sample properly. Keep the sample in a freezer at -20°C or -80°C until extraction. Freezing slows down enzymatic degradation of the DNA.

Common Mistakes

• Skipping the UV decontamination of tools and surfaces. UV is one of the most effective ways to destroy modern DNA, but it only works if the surface is clean and the UV lamp is not blocked by clutter.
• Using the same gloves for multiple samples. Even if you think you didn’t touch anything, gloves pick up DNA from the air and surfaces. Change them between every sample.
• Not wearing a proper face mask. A surgical mask may not be sufficient; an N95 or higher is recommended because it filters more particles. Also, ensure the mask fits snugly around the nose.
• Sampling the outer surface of the bone. The exterior is the most contaminated part. Always remove the outer layer and sample the interior.
• Over-UV-ing the sample. UV can damage ancient DNA, so limit exposure to the recommended time. If you are unsure, test a small piece first.
• Not running negative controls. Without a blank, you cannot tell if contamination occurred during extraction or sequencing.

Tips

• Work in a dedicated aDNA lab that is physically separated from modern DNA labs. The aDNA lab should have positive air pressure so that air flows out, not in, preventing outside particles from entering.
• Use a laminar flow hood or a PCR hood with HEPA filtration and UV light for extra protection.
• Keep the number of people in the lab to a minimum. Each person is a source of contamination.
• If possible, sample in a cold room or at least keep samples on ice to slow down DNA degradation.
• For tooth samples, the cementum layer (the outer layer of the root) is often rich in DNA. If you are targeting tooth, consider drilling into the root rather than the crown.
• Always use certified DNA-free tubes and pipette tips. Even if they are labeled sterile, they may contain DNA.

Troubleshooting

What if you see contamination in your negative control? This means contamination occurred during extraction or sequencing. Review your protocol: did you change gloves often enough? Did you use a fresh aliquot of reagents? Did you open the tubes in a clean area? You may need to repeat the extraction with new reagents and stricter practices.

What if your sample yields no DNA at all? This could be due to ancient DNA degradation, but it could also be that the UV treatment was too harsh or you sampled a bone region with low DNA preservation. Try a different part of the bone, such as the petrous bone (in skulls) which is known for better preservation. Also, check that your extraction protocol is optimized for aDNA.

What if your results show a mix of ancient and modern DNA? This is a sign of contamination during sampling or extraction. Check your negative controls. If they are clean, the contamination may have occurred before you received the sample (e.g., during excavation). In that case, you can try to bioinformatically separate the sequences, but it’s better to re-sample if possible.

What if your tools are not sterile? Always autoclave or sterilize tools before use. If you are in the field, use disposable sterile scalpels and forceps. Never reuse a blade.

What if the UV lamp is not working properly? UV bulbs lose efficiency over time. Replace them regularly (usually every 6-12 months) and test with a UV meter. If you suspect UV is not working, use a chemical decontamination method: soak tools in 10% bleach for 10 minutes, then rinse with sterile water and 70% ethanol.

Frequently Asked Questions

Q: Can I use regular gloves instead of DNA-free gloves?
A: Regular gloves may have DNA on them. Always use gloves that are certified DNA-free, and change them often. Even DNA-free gloves can pick up DNA from surfaces, so the key is to change them between samples.

Q: Is it okay to sample in the field, or should I wait until I’m in the lab?
A: Field sampling is possible but riskier. If you must sample in the field, wear gloves, use sterile tools, and immediately place the sample in a sterile tube. However, it’s best to bring the entire specimen to the lab where you can decontaminate the exterior properly.

Q: How long can I store a sample before extraction?
A: Ideally, extract as soon as possible. If you must store, keep it at -20°C or -80°C in a dry, sterile tube. Avoid freeze-thaw cycles, as they can damage DNA.

Q: What is the difference between a negative control and a blank?
A: They are often used interchangeably. Both are samples that contain no DNA and are processed alongside real samples to detect contamination. The negative control goes through all steps, from extraction to sequencing.

Q: Why do I need to use 10% bleach and 70% ethanol?
A: Bleach destroys DNA by breaking the strands, while ethanol removes organic residues and helps the bleach work better. They are used together to ensure surfaces are free of both DNA and other contaminants.

Q: Can I use a regular PCR hood instead of a dedicated aDNA lab?
A: A PCR hood can be used if it is in a clean, low-traffic area and has UV light. However, it is not a substitute for a dedicated aDNA lab, which has controlled air pressure and separate workflows for pre- and post-PCR steps.

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