Introduction

Forensic DNA laboratories routinely process evidentiary samples that vary widely in DNA quantity, sample quality, and profile complexity. STR systems used in casework must therefore deliver interpretable results across diverse sample types while maintaining efficiency and consistency within established workflows. To support casework requiring greater discriminatory power, expanded autosomal STR systems must deliver robustness, clear analytical interpretability, and ease of implementation, alongside increased marker content.

The VersaPlex® 31P System (Cat. No. DC8020) is a casework-focused 8-color STR multiplex that expands the autosomal STR marker set within a single-reaction workflow suitable for forensic laboratory operations. It was designed for compatibility with the Spectrum CE System and Spectrum Compact CE System.

The VersaPlex® 31P System includes the twenty Combined DNA Index System (CODIS) core loci, Penta D, Penta E, D6S1043, and six additional autosomal STR loci (D6S477, D3S3045, D19S253, D10S1435, D15S659, and D8S1132). The multiplex also incorporates the biological sex markers, Amelogenin and DYS391, along with two internal quality indicators, Quality Indicator Small (QIS) and Quality Indicator Large (QIL). The locus selection increases the number of independent autosomal markers available for profile comparison and contributor differentiation in evidentiary samples while maintaining a single-reaction workflow.

Amplicon size distribution was a key consideration in the multiplex design. Sixteen autosomal STR loci generate amplicons less than 250 bases in length, supporting allele recovery from degraded or fragmented DNA (Figure 1). By leveraging the expanded 8-dye capacity of the Spectrum CE Systems, increased locus content is achieved while maintaining adequate spacing between markers within individual dye channels, facilitating data analysis using standard forensic genotyping software.

DNA amplification with the VersaPlex® 31P System was designed for efficient processing of forensic casework samples while maintaining reliability. The system uses a single 25 µl amplification reaction optimized for a target template amount of 0.5 ng, with PCR amplification completed in under 80 minutes.¹,²

Performance Summary

Performance of the VersaPlex® 31P System was characterized under defined laboratory conditions. Donor-derived samples were collected from consenting individuals in compliance with Promega’s human subjects policy.

These studies were conducted to evaluate system behavior relevant to reliability and robustness under conditions consistent with forensic laboratory practice, using a variety of DNA samples that represent typical sample type and DNA quality. Across these studies, the VersaPlex® 31P System consistently generated interpretable STR profiles using the recommended amplification conditions.

Using a target template amount of 0.5 ng with the recommended amplification protocol, 2800M Control DNA produced reproducible STR profiles with balanced peak heights across all loci. Separation and detection were performed on the Spectrum CE System, and data were analyzed using GeneMarker®HID Software for Spectrum CE Systems.³ A representative electropherogram is shown in Figure 2.

Forensic casework often requires analysts to assess sample quality alongside STR profile interpretation. To support interpretation of profiles generated across this range of sample conditions, the VersaPlex® 31P System incorporates two internal quality indicators, QIS and QIL, which span the lower and upper size ranges of the multiplex. These indicators are co-amplified within each reaction and provide information regarding amplification success across the fragment size range. Differential behavior of the quality indicators provides context for interpretation; PCR inhibition is reflected by reduced amplification of the larger QI indicator, whereas degradation of the evidence sample DNA template does not impact QI indicator amplification. Inclusion of these quality indicators within the multiplex provides sample-level quality information to support casework interpretation without requiring additional reactions or workflow modifications.

A representative casework-type sample was generated from a bone specimen extracted using the Bone Extraction Kit followed by purification with the Maxwell® FSC DNA IQ Casework Kit on the Maxwell® RSC 48 Instrument.⁴-⁶ DNA quantification using the PowerQuant® System indicated DNA degradation, with an elevated [Auto]/[Deg] ratio greater than 2 ([Auto]/[Deg] = 4.71), but no evidence of PCR inhibition.⁷ Amplification was performed using a target template amount of 0.5 ng. A full biological male autosomal STR profile was obtained, with QIS and QIL exhibiting similar peak heights (Figure 3). The reduced peak heights observed at larger loci in the bone sample are consistent with the elevated [Auto]/[Deg] ratio and the presence of DNA degradation indicated by quantification.