Developmental validation of a custom-designed Multi-InDel panel: A five-dye multiplex amplification system for challenging DNA samples

The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study, we have introduced a custom-designed panel capable of simultaneously amplifying 41 novel Multi-insertion/deletion (Multi-InDel) markers and an amelogenin locus using the capillary electrophoresis platform. Through a developmental validation study conducted in accordance with guidelines recommended by the Scientific Working Group on DNA Analysis Methods, we demonstrated that the new Multi-InDel system exhibited the sensitivity to produce reliable genotyping profiles with as little as 62.5 pg of template DNA. Accurate and complete genotyping profiles could be obtained even in the presence of specific concentrations of PCR inhibitors. Furthermore, the maximum amplicon size for this system was limited to under 220 bp in the genotyping profile, resulting in its superior efficiency compared to commercially available short tandem repeat kits for both naturally and artificially degraded samples. In the context of mixed DNA analysis, the Multi-InDel system was proved informative in the identification of two-person DNA mixture, even when the template DNA of the minor contributor was as low as 50 pg. In conclusion, a series of performance evaluation studies have provided compelling evidence that the new Multi-InDel system holds promise as a valuable tool for forensic DNA analysis.