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Genetic Tools and Techniques in Forensic Science—an In-Depth Look at...
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Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Technologies and Resources for Genetics".

Deadline for manuscript submissions: 15 February 2025 | Viewed by 10126

Special Issue Editor

Dr. Ugo Ricci

E-Mail Website
Guest Editor
SOD Diagnostica Genetica, Azienda Ospedaliero Universitaria Careggi, 50134 Florence, Italy
Interests: forensic genetics; Italian NDA database; ISO/IEC 17025:2017; validation studies; short tandem repeats; capillary electrophoresis; DNA quantification

Special Issue Information

Dear Colleagues,

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process. In real casework, a trace may not always contain human biological material, rather the biological traces at crime scenes may not be of human origin. Furthermore, the amount of DNA in a sample can vary between tens or hundreds of nanograms. Quantification guarantees maximum efficiency and avoids repeated analyses, overamplified samples or unnecessary examinations.

This Special Issue is dedicated to the classic and latest quantitative techniques, studying the status, role, and future of quantification in forensic genetics. We also look forward to receiving other related papers.

Dr. Ugo Ricci
Guest Editor

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Keywords

  • forensic genetics
  • ISO/IEC 17025:2015
  • accreditation
  • internal validation
  • contamination prevention
  • risk management
  • quality control

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Published Papers (7 papers)

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Research

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15 pages, 1588 KiB  
Article
A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA
by Miriam Foster, Jennifer A. McElhoe and Mitchell M. Holland
Genes 2024, 15(9), 1129; https://doi.org/10.3390/genes15091129 - 27 Aug 2024
Viewed by 1090
Abstract
To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can [...] Read more.
To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10−8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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14 pages, 645 KiB  
Article
An Assessment of the Performance Limitations of the Integrated QuantifilerTM Trio-HRM Assay: A Forensic Tool Designed to Identify Mixtures at the Quantification Stage
by Chastyn Smith, Sarah J. Seashols-Williams, Edward L. Boone and Tracey Dawson Green
Genes 2024, 15(6), 768; https://doi.org/10.3390/genes15060768 - 12 Jun 2024
Viewed by 951
Abstract
Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated [...] Read more.
Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated QuantifilerTM Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated QuantifilerTM Trio-HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated QuantifilerTM Trio-HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor’s sex. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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17 pages, 4507 KiB  
Article
Looking into the Quantification of Forensic Samples with Real-Time PCR
by Ugo Ricci, Dario Ciappi, Ilaria Carboni, Claudia Centrone, Irene Giotti, Martina Petti, Brogi Alice and Elisabetta Pelo
Genes 2024, 15(6), 759; https://doi.org/10.3390/genes15060759 - 9 Jun 2024
Viewed by 1761
Abstract
The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA [...] Read more.
The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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14 pages, 1366 KiB  
Article
The Usefulness of qPCR Data for Sample Pre-Assessment and Interpretation of Genetic Typing Results
by Martina Onofri, Simona Severini, Federica Tommolini, Massimo Lancia, Cristiana Gambelunghe, Luigi Carlini and Eugenia Carnevali
Genes 2024, 15(6), 744; https://doi.org/10.3390/genes15060744 - 5 Jun 2024
Viewed by 967
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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14 pages, 2200 KiB  
Article
Assessing DNA Degradation through Differential Amplification Efficiency of Total Human and Human Male DNA in a Forensic qPCR Assay
by Elena Chierto, Serena Aneli, Nicola Nocco, Alessia Riem, Martina Onofri, Eugenia Carnevali and Carlo Robino
Genes 2024, 15(5), 622; https://doi.org/10.3390/genes15050622 - 14 May 2024
Cited by 4 | Viewed by 1693
Abstract
The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of [...] Read more.
The assessment of degradation is crucial for the analysis of human DNA samples isolated from forensic specimens. Forensic quantitative PCR (qPCR) assays can include multiple targets of varying amplicon size that display differential amplification efficiency, and thus different concentrations, in the presence of degradation. The possibility of deriving information on DNA degradation was evaluated in a forensic qPCR assay not specifically designed to detect DNA fragmentation, the Plexor HY (Promega), by calculating the ratio between the estimated concentrations of autosomal (99 bp) and Y-chromosomal (133 bp) targets (“[Auto]/[Y]”). The [Auto]/[Y] ratio measured in 57 formalin-fixed, paraffin-embedded samples was compared to a quality score (QS) calculated for corresponding STR profiles using quantitative data (allele peak height). A statistically significant inverse correlation was observed between [Auto]/[Y] and QS (R = −0.65, p < 0.001). The [Auto]/[Y] values were highly correlated (R = 0.75, p < 0.001) with the “[Auto]/[D]” values obtained using the PowerQuant (Promega) assay, expressly designed to detect DNA degradation through simultaneous quantification of a short (Auto) and a long (D) autosomal target. These results indicate that it is possible to estimate DNA degradation in male samples through Plexor HY data and suggest an alternative strategy for laboratories lacking the equipment required for the assessment of DNA integrity through dedicated qPCR assays. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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Review

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15 pages, 887 KiB  
Review
Genomic Multicopy Loci Targeted by Current Forensic Quantitative PCR Assays
by Richard Jäger
Genes 2024, 15(10), 1299; https://doi.org/10.3390/genes15101299 - 5 Oct 2024
Viewed by 1287
Abstract
Modern forensic DNA quantitation assays provide information on the suitability of a DNA extract for a particular type of analysis, on the amount of sample to put into the analysis in order to yield an optimal (or best possible) result, and on the [...] Read more.
Modern forensic DNA quantitation assays provide information on the suitability of a DNA extract for a particular type of analysis, on the amount of sample to put into the analysis in order to yield an optimal (or best possible) result, and on the requirement for optional steps to improve the analysis. To achieve a high sensitivity and specificity, these assays are based on quantitative PCR (qPCR) and analyze target DNA loci that are present in multiple copies distributed across the genome. These target loci allow the determination of the amount of DNA, the degree of DNA degradation, and the proportion of DNA from male contributors. In addition, internal control DNA of a known amount is analyzed in order to inform about the presence of PCR inhibitors. These assays are nowadays provided as commercial kits that have been technically validated and are compatible with common qPCR instruments. In this review, the principles of forensic qPCR assays will be explained, followed by information on the nature of DNA loci targeted by modern forensic qPCR assays. Finally, we critically draw attention to the current trend of manufacturers not to disclose the exact nature of the target loci of their commercial kits. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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Other

Jump to: Research, Review

13 pages, 1165 KiB  
Brief Report
Utilization of qPCR to Determine Duration and Environmental Drivers Contributing to the Persistence of Human DNA in Soil
by Hannah L. Noel, Rebecca L. George, Brittania Bintz, Maureen Peters Hickman and Frankie West
Genes 2024, 15(6), 741; https://doi.org/10.3390/genes15060741 - 5 Jun 2024
Viewed by 1588
Abstract
Little is known about the underlying mechanisms that contribute to the persistence and degradation of DNA within soil. The goals of this study are to determine the duration of mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) persistence in soils enriched by surface-level human [...] Read more.
Little is known about the underlying mechanisms that contribute to the persistence and degradation of DNA within soil. The goals of this study are to determine the duration of mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) persistence in soils enriched by surface-level human decomposition and to better understand the contribution of environmental factors. The surface-level decomposition of three human cadavers was documented over 11 weeks. Based on quantitative PCR results, we found nuDNA to persist in soils six weeks post-placement, while mtDNA was recoverable for the entire 11-week decomposition period. Principle components analyses and Spearman’s rank correlations revealed that (1) time, (2) total body score, and (3) weekly average air temperature were significantly correlated with concentrations of nuDNA and mtDNA in soil, suggesting these factors play a role in the degradation of DNA in soils. Full article
(This article belongs to the Special Issue Genetic Tools and Techniques in Forensic Science—an In-Depth Look at the Process of Quantification of Forensic Samples)
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