Does zero really mean nothing?—first experiences with the new PowerQuantTM system in comparison to established real-time quantification kits

Abstract

DNA quantification is an important step in the molecular genetic analysis of a forensic sample, hopefully providing reliable data on DNA content for a subsequent generation of reproducible STR profiles for identification. For several years, this quantification has usually been done by real-time PCR protocols and meanwhile a variety of assays are commercially available from different companies. The newest one is the PowerQuant(TM) assay by Promega Inc. which is advertised with the promise that a determined DNA concentration of 0 ng/μl in a forensic sample guarantees the impossibility to achieve true STR results, thus allowing to exclude such samples from STR analysis to save time and money. Thus, the goal of this study was to thoroughly verify the quantification step with regard to its suitability as a screening method. We have evaluated the precision and reliability of four different real-time PCR quantification assays by systematically testing DNA dilutions and forensic samples with various DNA contents. Subsequently, each sample was subjected to the Powerplex® ESX 17 fast kit to determine a reliable cutoff level for exclusion of definitely negative samples from STR analysis. An accurate quantification of different cell line DNA dilutions was not possible with any kit. However, at least the PowerQuant(TM) assay provided suitable data analyzing forensic samples, whereas in other systems up to 46 % of negative samples still displayed reliable STR analysis results. All in all, the PowerQuant(TM) assay represents a big step forward, but the evaluation of real-time PCR quantification results has still to be done with great care.

CSYseq: The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics

“…Although this statement needs to be confirmed by future studies with more challenging samples, this is in accordance to observations made by Poetsch et al . [ 62 ]. They concluded that only 0.00 ng/μl indicates the absence of DNA with the PowerQuant kit, which demonstrates that a concentration of >0.01 ng/μl could still provide a full Y-STR profile after multiplex PCR and CE.…”

Development of a quantitation approach for total human and male DNA based on real time PCR followed by high resolution melting analysis

“…However, Poetsch et al. described that the comparability of quantification results between different kits does not seem to be possible due a greater variance of results found by analyzing serial dilutions of standard DNA.…”

“…Furthermore, Poetsch et al. reported the comparison among four human DNA quantification kits (Plexor, Quantifiler, Quantiplex and PowerQuant) and they described that full or partial profiles could be found in samples where their quantification value was under the threshold line. Therefore, failures in reliability are found even in commercial kits where complexity and costs exceeds those compared home‐made techniques such as Amel‐Y duplex.…”

About the influence of environmental factors on the persistence of DNA — a long-term study

“…DNA extraction was performed using DNA IQ Casework Pro Kit and Casework Extraction Kit in the Maxwell 16® instrument according to the manufacturer’s instructions (Promega, Mannheim, Germany), resulting in an extraction volume of 50 µl. DNA concentration of samples was established by Real-time PCR using the PowerQuant™ System (Promega) according to the manufacturer’s instructions providing a reproducible and reliable detection threshold at least down to 25 pg DNA [ 23 ]. Using 2 µl DNA-containing solutions each sample was analyzed in duplicates.…”