Establishing a comparable test setup

In our laboratory, DNA is routinely isolated from skin flakes using the ReadyAmp™ Genomic DNA Purification System (Promega, Madison, WI, USA). To create the STR profiles, amplifications are carried out using the PowerPlex® ESX 17 Fast and ESI 17 Fast Systems in a reduced PCR volume of 14 µl in a 32-cycle PCR (in-house validation for skin flakes and single cells) [2, 3]. Separation of amplicons according to fragment length is performed on a 3500xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Data analysis was carried out using the GeneMapper® ID-X Software (Thermo Fisher Scientific, Waltham, MA, USA). According to the manufacturer’s information, the PCR setup of the PowerPlex® 18E, can be carried out in a 25 or 12,5 µl reaction volumes.

Since eight-colour chemistry cannot currently be measured on am ABI 3500xl Genetic Analyzer, fragment length determination was performed on a Spectrum Compact CE System (Promega, Madison, WI, USA) and data analysis was carried out using GeneMarker®HID V3.2.0 for Spectrum CE Systems software (SoftGenetics, State College, Pennsylvania, USA). In order to create comparable initial conditions for testing PowerPlex® 18E vs. PowerPlex® ESX/ESI 17 Fast Systems and ultimately “not comparing apples to oranges”, the following questions must be clarified in advance and appropriate validations must be carried out:

Do the two capillary electrophoresis devices show comparable sensitivity?

Validation: PowerPlex® ESX 17 Fast amplicons of a dilution series (500–12.5 pg DNA) as well as six replicates of samples containing 12.5 pg DNA were analysed on the ABI 3500xl (standard settings 1,2 kV und 24 sec) as well as on the Spectrum Compact CE System (measurement settings according to the manufacturer’s instructions 1,6 kV und 9 as well as with increased injection times of 15 and 25 seconds). The average peak heights and the number of dropouts per sample were determined (analytical threshold 100 rfu).

Result: To ensure comparable sensitivity of the two measuring systems, the injection time on the Spectrum Compact CE system must be increased to at least 15 seconds.

What impact does reducing the reaction volume from 25 to 14 µl have on the quality of the PowerPlex® 18E System results?

Validation: DNA dilution series (500–12.5 pg) were amplified using PowerPlex® 18E System in reaction volumes of 25 or 14 µl. The respective amplicons were measured with three different injection times (5, 15 and 25 seconds) on the Spectrum Compact CE system.

Result: Across all dilution levels, on average there are higher peaks when amplification was carried out in the smaller reaction volume of 14µl (Fig. 3). Moreover, with increasing DNA input (50, 25 and 12.5 pg DNA) fewer dropouts (or peaks below the analytical threshold of 100 rfu) can be observed (Fig. 4). Reduction in reaction volume therefore leads to improvement in profile quality. While increasing the injection time also leads to higher average peak heights across all dilution levels, the number of dropouts remains constant at dilution levels 25 and 12.5 pg with an injection time of 15 and 25 seconds, respectively. One explanation for this is that these are “real” dropouts, caused by stochastic effects, and not just peaks under 100 rfu that can be pulled above the analytical threshold by increasing the injection time. Because increasing injection time also favoured the origin of spikes, we decided on a injection time of 15 seconds.