Forensic DNA analysts have relied on the ease and accuracy of short tandem repeat (STR) genotyping technology for almost 30 years. The increased use of STR kits is accompanied by the growth in DNA databases over the years, resulting in the inevitable increase of potential adventitious matches with the older smaller STR panels. To reduce this undesired potential, larger panels of STR markers started to be used. These large STR panels can result in some performance limitations with challenging samples, as they use a broad range of amplicon sizes. With detection of only six dyes using a standard capillary electrophoresis (CE) instrument, an amplicon size range of approximately 60 to 500 bp is necessary to include all the required loci. The use of long amplicons becomes problematic while genotyping highly degraded DNA, as commonly recovered from a crime scene, because they would be the first to “drop out.” Additionally, these longer amplicons would also be more likely to drop out in the presence of amplification inhibitors. Consequently, the loci with long amplicons provide less information with challenging forensic samples.

To address the limitation of 6-dye CE technology, Promega developed a new 8-dye CE instrument, the Spectrum CE System, and an accompanying 8-dye STR amplification system, the PowerPlex® 35GY System, which co-amplifies the most markers in one amplification than any other commercial STR kit. The 35 markers in this STR multiplex system include 23 autosomal loci which satisfies the CODIS and ENFSI requirements, 11 Y-STR loci, and Amelogenin (Figure 1).

Figure 1: The PowerPlex® 35GY System - Next Generation STR Multiplex

With two new dye channels, the median allele size of autosomal loci in PowerPlex® 35GY System is 25% smaller than the median allele size in 6-dye autosomal STR systems. Additionally, alleles for 22 autosomal loci in PowerPlex® 35GY System are smaller than 325 bp, reducing the occurrence of dropout and providing maximal information from amplification of degraded or inhibited samples. Furthermore, the most discriminatory STR locus, SE33, has the smallest panel in this kit that in any other commercial STR kit, thereby improving the discrimination power of partial profiles.

In addition to smaller autosomal STR amplicons, this 8-dye STR multiplex also amplifies 11 Y-STR loci, making it the largest CE-based commercial STR multiplex system. To provide the most information from each crime scene sample, the Y-STR loci are selected based on their high gene diversity, which also increases the potential of more accurately identifying the minimal number of male contributors in a mixture (Figure 2). Additionally, the Y-STRs could provide an opportunity to simplify the familial DNA searching process. Currently, familial searching is a two-step process. The first step is to search for potential male relatives of the offender in an autosomal database. The second is to exclude false potentials by matching Y-STR haplotypes. Data from PowerPlex® 35GY System allows direct, one-step searching using Y-STR haplotypes.

Figure 2: A 2-person Male:Male Mixture Genotyped with PowerPlex® 35GY System

To further improve the ease-of-use of this trusted CE technology, the PowerPlex® 35GY System has two Quality Indicators, QIS and QIL, as the smallest and largest peak, respectively, in the blue channel. The QIL is designed to be sensitive to commonly found PCR inhibitors and could confirm indication of inhibitors detected during quantification. When direct amplification of single source reference and database samples are performed without DNA quantification, where there would be no indication of inhibitor presence, sloping STR peaks accompanied by low QIL peak will indicate inhibition of the amplification reaction (Figure 3).

Figure 3: Quality Indicators – QIL peak height is reduced in the presence of PCR inhibitors

However, sloping STR peaks accompanied by high QIL peak indicate that the DNA is degraded (Figure 4). The ability to distinguish inhibited samples from degraded samples by the low or high QIL peak heights helps DNA analysts determine the next best course of action for the analysis, thus saving sample, time, and cost.

Figure 4: Quality Indicators – QIL peak height is not affected in the presence of degraded DNA

This 8-dye STR system, together with the Spectrum CE system, has the potential to generate more information from each forensic crime scene and database sample than 6-dye CE-based assays. In addition to significantly enhancing the quality of STR analysis with 8-dye technology, the Spectrum CE system was designed to improve efficiency with simplified sample setup and operation. It has four 96-well plate positions that are continuously accessible, allowing DNA analysts to load and unload plates while the instrument is running. This will improve the productivity of the forensic laboratory by allowing partially full plates to be run and reducing instrument scheduling conflicts without sacrificing time or efficiency. Rush cases can be prioritized to run first even when the plate was added to the queue later. Additionally, four plate positions will increase overnight and weekend throughput without increasing the number of CE instruments.