Introduction

Capillary electrophoresis (CE) technology remains the standard in forensic DNA laboratories for producing reliable data in a cost-effective and timely manner. The new Spectrum CE System builds on the CE tradition of 4-, 5- and 6-color separation and offers an expanded spectral capacity to allow for the separation of eight color channels when used with the PowerPlex® 35GY System.

The Power of 8-Dye Chemistry

The expansion of the number of Combined DNA Index System (CODIS) core loci in the United States from 13 to 20 short tandem repeat (STR) loci was designed to reduce the potential for adventitious matches occurring within the dataset, to increase international compatibility for data sharing and to increase discrimination power in missing persons cases. Moving from 5- to 6-dye multiplexes enabled the increase to 20, but most in the community would agree that any future loci additions would require them to be larger amplicons, making them more difficult to consistently amplify in a 6-dye multiplex system with limited space. Shifting those additional markers into two new dye channels allows more loci to be included, providing more information from a sample and improving analyses. Moreover, in an 8-dye multiplex system, the narrow range of amplicon lengths enables more consistent results and allows for more mini-STRs to provide better results with degraded samples.

The PowerPlex® 35GY System allows co-amplification and fluorescent detection of the 20 CODIS Core Loci plus Amelogenin and DYS391 for gender discrimination. Penta D, Penta E and SE33 also are included to increase discrimination and allow searching of databases that contain profiles with these loci. With the two additional colors for detection, there are a total of 15 autosomal loci smaller than 250bp, aiding the analysis of difficult samples. Additionally, we’ve designed the layout to allow smaller amplicon sizes for these particularly informative loci: SE33 and D2S1338. Among commercially available kits, the PowerPlex® 35GY System has the smallest SE33 amplicon.

The insertion of Y-STR loci along with the autosomal loci increases the amount of genetic information obtained in a single amplification and can provide valuable data for familial searching or it can assist in the analysis of sexual assault evidence by helping to better determine the number of male contributors in mixed samples.

Figure 1. Configuration of the PowerPlex® 35GY System. The PowerPlex® 35GY System allows co-amplification and detection of 35 loci (24 autosomal and 11 Y-STR loci): CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433 and D22S1045, SE33, Penta D, Penta E and Amelogenin; DYS391, DYS385a/b, DYS390, DYS438, DYS448, DYS456, DYS458, DYS481, DYS635 and DYS643. Two Quality Indicators (QIS and QIL) are also included to assist with data interpretation.

Quality Indicators Provide Additional Insight for Data Interpretation

As workloads have increased, the desire to gain more information with the multiplex reaction has also increased. Samples and available time are precious enough without having to repeat STR analyses that failed to yield a full profile due to inhibition or degradation. To help you better interpret the quality of your STR results, the PowerPlex® 35GY System is the first PowerPlex® system to include two Quality Indicators, QIS and QIL.

The Quality Indicators can take the guesswork out of determining whether the amplification was successful by confirming if there is DNA present or not. Additionally, they can indicate inhibition that was not observed during the quantification step. Lastly, knowing whether the sample was inhibited or degraded enables you to make an informed decision about reworking the sample.

In Figure 2, Panel A shows a successful STR profile with balanced QIS and QIL peaks. Panel B shows no STR peaks but has balanced QIS and QIL peaks, indicating that no template DNA was amplified in the sample. Panel C is an example of a sloping STR profile with balanced QIS and QIL peaks, signaling that the sample is degraded. Panel D is a sloping STR profile but with a low or missing QIL peak on the right. This suggests that the sample is inhibited.

In cases where the electropherogram shows that neither Quality Indicator peak is present, then amplification has failed.

Figure 2. Quality Indicators for the PowerPlex® 35GY System. Balanced QIS and QIL peaks indicate a succesful STR profile (Panel A). An STR profile with balanced QIS and QIL peaks but no STR peaks indicates that no template DNA was amplified (Panel B). A sloping STR profile with balanced QIS and QIL peaks signals degraded sample (Panel C) while a sloping STR profile with a low or missing QIL peak suggests the sample is inhibited (Panel D).

One Amplification Kit for Casework and Databasing

The PowerPlex® 35GY System was designed for amplification of extracted DNA and direct amplification of single-source reference samples, enabling laboratories to process all sample types with one amplification kit. The PowerPlex® 35GY System is optimized to work with 1ng of extracted DNA in a 25µl reaction volume (Figure 3).

Figure 3. Amplification of extracted DNA with the PowerPlex® 35GY System. Extracted DNA (1ng) was amplified in a 25µL reaction volume to yield an 8-channel STR profile.

Excellent Results with Reduced Volume Reactions

As with our previous PowerPlex® Systems, the PowerPlex® 35GY System enables full and half volume direct amplification reactions. In the run below (Figure 4), DNA from a buccal swab in SwabSolution™ reagent was amplified for 25 cycles in a 12.5µl reaction volume. Amplified samples were separated on the Spectrum CE System.

Figure 4. Reduced volume amplification reaction with the PowerPlex® 35GY System. DNA from a buccal swab sample was directly amplified for 25 cycles in a 12.5µL reaction volume.

More Data with Low DNA Input

While the PowerPlex® 35 GY System has been optimized to work with 1ng of extracted DNA, the kit also performs well with smaller amounts of input DNA. Because the total number of loci is higher in the PowerPlex® 35GY System than in other commercially available kits, the total number of alleles will be higher as well, providing more useful results to casework laboratories. The chart below indicates the average number of alleles and the percentage of alleles called with various amounts of input DNA (Figure 5).

Figure 5. More loci provide more data even with low DNA input. The average number of alleles and the percentage of alleles called with varied DNA input amounts is shown for samples amplified with the PowerPlex® 35GY System and separated on the Spectrum CE System.

Robust Performance in the Presence of Strong Inhibitors

Inhibitors can interfere with PCR amplification, reduce PCR efficiency or even cause complete amplification failure. The superior buffer system included in the PowerPlex® 35GY System ensures strong performance even with the most inhibited samples. The kit minimizes the need to re-amplify samples generally thought to be too challenging to run, saving your lab hours of repeat analyses.

More Mini-STRs for Improved Success with Difficult Samples

The varied environmental conditions encountered in forensic casework, mass disasters and missing persons scenarios can lead to severe degradation of human DNA samples. Such highly degraded samples often produce incomplete or no STR profiles due to larger loci that often “drop out”. The inclusion of more loci as mini-STRs in the PowerPlex® 35GY System results in better and more complete profiles, making it an ideal option to process cold case samples and newer, difficult sample types. For instance, in the PowerPlex® 35GY System, there are eight common loci whose amplicons are at least 100bp smaller in size than when amplified using the PowerPlex® Fusion 6C System.

More Successful Mixture Deconvolution

Casework mixture samples often contain PCR inhibitors, and the minor component may be present at a very low level. The enhanced sensitivity, superior capability to overcome inhibition and the inclusion of Y-STRs in the PowerPlex® 35GY System enables laboratories to analyze and interpret mixtures more easily and reliably than was previously possible (Figure 6).

Figure 6. Mixed sample analysis with the PowerPlex® 35GY System. STR profiles for mixed male-female samples are deconvoluted to provide the male-to-female DNA ratios present in the samples.

In summary, the 8-color PowerPlex® 35GY System offers several benefits over current 5- and 6-color systems, such as improved results from the most challenging samples, more reliable mixture deconvolution and more information from each analysis. The system works exclusively on the Spectrum CE Instrument. Together, these powerful tools enable advanced multiplexing to support your forensic and paternity workflow needs.