What happens to wildlife DNA after the evidence has degraded?

In wildlife forensic genetics, the question is not only whether DNA is present. A sample can contain DNA and still fail to answer the question an investigator needs answered. Degradation, substrate effects, processing, mixtures and marker choice all shape what can be interpreted.

Can the DNA that remains still answer the forensic question being asked?

That question runs through the project. Species identification, individualization and mixed-product detection are related, but they do not ask the same thing and they do not always need the same method.

One part of the research examined blesbok blood traces across surfaces and environments that better reflect imperfect evidence. 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.

Sampling continued for 102 days. The aim was not to create a perfect laboratory scenario, but to examine how target DNA behaves when blood evidence is exposed to time, surfaces and environmental conditions.

The research separates two forensic questions that are sometimes treated too loosely in public explanations. Identifying the species is not the same as identifying the individual animal. The methods overlap in purpose, but they are not interchangeable.

DNA barcoding can remain useful where nuclear marker recovery becomes weaker, but it answers a different forensic question. A species-level result can support identification of blesbok as the source species. It does not by itself identify which individual animal the sample came from.

Wildlife forensic evidence is not always a blood trace on a surface. It may be a processed product. Processing can fragment DNA, introduce inhibitors, alter recovery and make mixtures harder to interpret.

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 probe component 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.

This part of the work is therefore assay development, not a successfully validated forensic test. It highlights the importance of probe design, binding stability and validation before a targeted qPCR approach can be relied on for forensic interpretation.

1. 1. Mitochondrial DNA barcoding

   Used to answer the species-level question, particularly through the mitochondrial COI region.

2. 2. Microsatellite profiling

   Used to assess individual-level comparison potential through nuclear markers, with attention to locus and allele recovery as samples aged.

3. 3. DNA concentration and purity assessment using NanoDrop

   DNA concentration and purity were assessed using a NanoDrop spectrophotometer. This step described the extract; it did not prove which species or individual was present.

4. 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. 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.

No single method works for every sample. A result that is useful for species identification may not support individualization. A method that works for a single-species product may struggle with a mixed-species product. A sample that contains DNA may still fail to produce the target result needed for the forensic question.

Interpretation depends on:

• sample type
• age
• environmental exposure
• substrate
• processing
• mixture composition
• target marker
• forensic question

The main contribution of this research is not simply showing that DNA can survive degradation. It is identifying where current wildlife forensic techniques remain useful, where their limitations begin, and how the resulting evidence should be interpreted without overstating what the science can support.