Internal Validation of the PowerPlex® Y23 PCR Amplification System for Reduced Volume to Improve Cost Effectiveness in Forensic DNA Analysis

The PowerPlex® Y23 system is one of the widely used PCR amplification kits in forensic analysis of male specific DNA markers. The kit amplifies commonly available 17 Y-STR and 6 newly incorporated Y-STR loci providing enhanced discrimination power for male lineage identification. The system's high sensitivity makes it especially beneficial in forensic applications such as sexual assault investigations, paternity testing, kinship analysis and a range of other forensic applications. The PowerPlex Y-23 kit was previously validated in the Government Analyst’s Department's DNA laboratory, following the manufacturer's recommendations for full-volume (25µl) reactions, establishing a working range of 0.25ng to 0.5ng. To reduce the annual chemical costs, an internal validation was performed using a reduced reaction volume of 12.5µl. The internal validation evaluated the system's sensitivity, repeatability, reproducibility, mixture analysis, and contamination assessment through the amplification of received male DNA samples from a case study and male standard 2800M of PowerPlex® Y23 system. Sensitivity studies revealed the optimal template DNA input working range for achieving a complete profile was 0.25ng to 0.5ng. The system's repeatability and reproducibility also confirmed consistent results across repeated studies. Additionally, the mixture analysis of two male DNA samples at 1:1 ratio successfully detected all alleles contributed by both individuals. However, when the ratio becomes 1:3 the allele dropout starts. The study confirms that the amplification system is a robust, reliable, and cost-effective solution for Y-chromosome DNA profiling, even when using a reduced reaction volume of 12.5 µl, making it highly suitable for forensic applications.