Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System.

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results.

Recognizing the advantage of employing the PowerPlex(®) ESI and PowerPlex(®) ESX complementary kits for purposes of clarification in routine casework.

In an effort to promote European cross-border cooperation in fighting crime and international terrorism the Treaty of Prüm was drafted and accepted within the European forensic community. This move led to the commercial development of new multiplex kits which introduced five new STR loci and promised better performance. Recently the Israel Police DNA Casework and Database laboratories adopted the PowerPlex(®) ESI kit for routine use in our laboratories. Presented in this paper are examples of three types of ambiguous results encountered during the implementation of the PowerPlex(®) ESI kit into routine work. These ambiguous products presented themselves in the form of (1) extreme variants outside of loci borders, (2) failure to amplify sister allele pairs or expression of null alleles and (3) episodes of loss of separation of adjacent microvariants primarily in mixture samples. The re-analysis of all these samples using the PowerPlex(®) ESX kit successfully and rapidly clarified all three categories of anomalies. Spotlighting such events to the forensic community, especially regarding the novel loci introduced in these next generation kits, can aid in raising the analyst's awareness to their future appearances and prevent possible erroneous conclusions. In addition, providing timely DNA results to investigating teams is of great importance and operational forensic laboratories do not have at their immediate disposal methods such as sequencing to elucidate such manifestations. We suggest that the complementary use of the PowerPlex(®) ESI and PowerPlex(®) ESX can provide a benefit for clarification purposes in routine casework.