Interpretation of mixed DNA samples remains a major challenge in forensic genetics, particularly when contributors differ in DNA integrity. Conventional STR-based workflows treat degradation primarily as a limiting factor rather than as an informative analytical feature and rely mainly on post hoc mathematical deconvolution. In capillary electrophoresis data, degradation is considered to reduce profile quality rather than assisting in contributor assessment. However, DNA from different contributors in a mixture typically differs in integrity, and this difference can be used as a basis for separation. Here, we present an experimental forensic approach for mixed DNA analysis based on degradation-dependent amplifiability across size-specific multiplex PCR assays.

  In this procedure, the DNA extracted from a mixed sample is divided into three aliquots, each of which is subjected to a separate multiplex PCR amplification. Each assay contains the complete spectrum of (STR) markers. All amplicons fall within defined size ranges—depending on the set—with short (<150 bp), medium (~300 bp), or long (~600 bp) PCR products. Resulting profiles are detected by nanopore sequencing.

Our results show that a contributor with degraded DNA can only be detected in short amplicon multiplex. DNA with high integrity, on the other hand, is detected in all three multiplexes. By combining the three datasets, meaningful amplification patterns can be identified. 

Model mixture experiments showed that combining size-specific amplification with vector-based software interpretation enables substantially clearer assignment of contributor alleles than conventional multiplex STR analysis. 

This strategy may improve forensic mixture interpretation in casework involving environmentally challenged samples, trace DNA, and anthropological material.

Authors

• Vladimir Orekhov (MaeSTRo Lab Oy, Finland)
• Antti Sajantila (University of Helsinki, Finland)
• Peter Dobrowolski (MaeSTRo Lab Oy, Finland)