What happens to wildlife DNA after the evidence has degraded?
Wildlife forensic evidence is rarely neat. It may be old, exposed to heat and weather, processed into a product, contaminated by its surroundings, or mixed with DNA from other species before it reaches a laboratory.
My MSc research looks at what happens to blesbok DNA under those conditions. The work separates environmental blood evidence from processed wildlife products, species identification from individual-level comparison, and DNA concentration or purity assessment from targeted qPCR assay development.
When does biological evidence stop being useful?
Recovering DNA is the first step. The condition of the sample, the surface, processing, mixtures and the chosen genetic marker determine which forensic questions the result can answer.
Can the DNA that remains still answer the forensic question being asked?
The project examines species identification, individual-level comparison and detection in mixed products. Each question requires suitable markers and careful interpretation.
Degraded blood evidence across realistic surfaces and environments
One part of the research examined blesbok blood traces on surfaces and in environmental conditions that may be encountered during an investigation. The tested substrates were metal, wood, rubberized car mat, cotton, soil and grass. The environmental conditions were indoors, semi-covered outdoors and fully exposed outdoors.
Tested substrates
- Metal
- Wood
- Rubberized car mat
- Cotton
- Soil
- Grass
Environmental conditions
- Indoors
- Semi-covered outdoors
- Fully exposed outdoors
Sampling continued for 102 days to examine how time, different surfaces and environmental exposure affect the recovery of target DNA from blood evidence.
Results summary
- Blesbok was identified from metal, wood, and car mat on days 1 and 7 across all conditions.
- Blesbok was identified from indoor samples after 51 days.
- Cotton produced no species-specific blesbok result.
- Soil and grass produced non-target species.
- DNA extraction was possible in samples that did not produce a species-specific blesbok result.
Species identification and individual-level comparison
Species identification asks which species the sample came from. Individual-level comparison asks whether a profile may be associated with a particular animal. These questions use different genetic information.
Mitochondrial DNA barcoding
- Answers the species-level question.
- Uses the mitochondrial COI region.
- Remained more robust in degraded samples.
Microsatellite profiling
- Can support individual-level comparison.
- Uses nuclear markers.
- Locus and allele recovery declined as samples aged.
Mitochondrial DNA barcoding remained useful in some samples where nuclear marker recovery was weaker. It supported species-level identification of blesbok, while microsatellite profiling was used for individual-level comparison.
What happens when wildlife products are processed or mixed?
Wildlife evidence may also be recovered from processed products. Processing can fragment DNA, introduce inhibitors, affect recovery and make mixtures harder to interpret.
Biltong
The biltong sample represented a single-species, whole-muscle product. It was processed with salt, vinegar, spices and other ingredients.
DNA barcoding identified blesbok in the biltong after seven months, showing that species-level DNA remained recoverable from this product.
Dry sausage
The dry sausage represented a mixed-species product. Sequences suitable for species identification were obtained, but only cattle, Bos taurus, was identified using standard DNA barcoding.
Blesbok was not detected using standard DNA barcoding, but that does not prove blesbok was absent. Mixture heterogeneity, dominant-template amplification and preferential amplification may have contributed to the result.
Developing a targeted method for mixed-species detection
qPCR was investigated to detect blesbok specifically within mixed dry sausage. That targeted assay work was separate from DNA concentration and purity assessment, which was performed using a NanoDrop spectrophotometer.
The assay included primers, a blesbok-specific probe and a mammalian probe. Primer screening was successful, which showed that the primer component could amplify as intended during screening.
The probes did not perform successfully. The probes were too short and lacked sufficient binding stability. That probe failure prevented successful probe-based detection and multiplex validation.
The assay remains under development and has not been validated as a forensic test. Further work is needed on probe design, binding stability and multiplex validation.
The techniques used
1. Mitochondrial DNA barcoding
The mitochondrial COI region was amplified and sequenced for species identification.
2. Microsatellite profiling
Used to assess individual-level comparison potential through nuclear markers, with attention to locus and allele recovery as samples aged.
3. DNA concentration and purity assessment using NanoDrop
DNA concentration and purity were measured using a NanoDrop spectrophotometer before further analysis.
4. Species-specific qPCR assay development
Investigated as a targeted method for detecting blesbok DNA within a mixed-species dry sausage sample. Primer screening was successful, but probe failure prevented full detection and multiplex qPCR validation.
5. Substrate and processing experiments
Used to examine how surfaces, environmental exposure, product processing and mixture composition affect the usefulness of wildlife forensic DNA evidence.
The result depends on the evidence and the question
Method choice depends on the sample and the forensic question. DNA barcoding can support species identification, while microsatellite profiling provides individual-level comparison when enough nuclear DNA is recovered. Mixed products may require targeted or mixture-sensitive methods.
Interpretation depends on:
- sample type
- age
- environmental exposure
- substrate
- processing
- mixture composition
- target marker
- forensic question
This research identifies where current wildlife forensic methods remain useful after environmental exposure or processing, where their limitations begin and how the results should be interpreted.
Why this matters for wildlife forensic investigations
Wildlife forensic laboratories often receive degraded, processed or mixed evidence. The chosen method and the interpretation of its result must fit the condition of the sample and the question being asked.
- Old blood traces may still retain useful species-level information.
- Indoor protection can extend target-DNA persistence.
- Seven-month-old biltong remained identifiable.
- Mixed products require targeted or mixture-sensitive approaches.
- A negative or non-target result does not automatically prove absence.