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. 2024 Jun 5;15(6):744.
doi: 10.3390/genes15060744.

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results

Martina Onofri  1   2 , Simona Severini  1 , Federica Tommolini  1 , Massimo Lancia  2 , Cristiana Gambelunghe  2 , Luigi Carlini  1   2 , Eugenia Carnevali  1
Affiliations

The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results

Martina Onofri et al. Genes (Basel). .

Abstract

DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.

Keywords: average peak height; casework samples; forensic genetics; human DNA quantification; probative STR profile; qPCR variation; trace samples.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
PowerQuant® System results for the [Auto] target of five serial three-fold dilutions. Above is the correlation of qPCR measurements with true concentration. Below, the boxplots show the median and distribution of the measurements for each dilution.
Figure 2
Figure 2
Histograms representing the distribution of peak heights across replicates for each dilution. On the y-axis, the numerosity of samples that fall within a peak height range is reported. The x-axis bin width was set to 80 RFU. The vertical black dashed line represents the overall mean peak height (RFU) calculated across all replicates, while the colored dashed lines represent the mean height of each replicate. (A) All dilutions; (B) zoomed-in view of dilutions B and C; (C) zoomed-in view of dilutions D and E.
Figure 3
Figure 3
Graphical representation of the peak heights (RFU) detected per each locus across all five 2800 M dilution series samples. Each point represents a single replicate. The height of the barplot falls at the mean height (RFU) calculated for each locus across replicates, while the error bar indicates the standard deviation.
Figure 4
Figure 4
Correlation of mean peak height versus DNA template concentration for the replicates of the control 2800 M control DNA dilution series (above) and of the casework samples (below). The correlation of mean peak height for casework samples with DNA template (continuous line) is compared with the regression line fitted on the 2800 M control DNA dilution series (dashed line).
See this image and copyright information in PMC

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