Identification of a novel SNP mutation causing drop-out alleles in a paternity test using combined nest and touch-down PCR with Sanger sequencing

Background: Short tandem repeat (STR) markers are widely used in forensic DNA analysis due to their ability to provide automated and standardised typing. However, incorrect STR typing can have a significant impact on forensic outcomes.

Aim: In this study, we detected drop-out alleles at the SE33 locus in a putative father-son pair using the Microreader^™ 28 A ID System. This result could lead to a false conclusion of non-paternity.

Subjects and methods: To investigate the cause of the drop-out alleles, we developed a nest and touch-down PCR program for Sanger sequencing of the SE33 locus. Subsequently, we investigated the mutation frequency in 300 unrelated individuals and reviewed the results of 429 paternity tests.

Results: The results showed that the frequency of the G > T mutation at this locus was less than 0.01, which is a novel and rare mutation. Our analysis revealed a novel G > T mutation in the primer-binding region of both samples, which was a rare single-nucleotide mutation site in the Chinese population. This variation was found to be responsible for the drop-out alleles observed in the samples.

Conclusion: Our findings have important implications for optimising primer design and constructing DNA databases for forensic analysis.