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Sviluppo e validazione di un saggio in real-time pcr per la determinazione della quantità e qualità del dna nucleare e mitocondriale umano e le sue applicazioni nelle analisi forensi
Full text linkLa quantificazione del DNA umano ha un ruolo molto importante nella genetica forense. Una stima quanto più accurata della quantità di DNA umano è indispensabile per una pianificazione e un’ottimizzazione delle reazioni di genotipizzazione, così come è altrettanto utile una valutazione della presenza di sostanze inibitrici della reazione di PCR presenti nei campioni forensi. Inoltre, per campioni altamente compromessi, la quantificazione può fornire informazioni sullo stato di degradazione del DNA, indirizzando l'analista forense verso strategie di genotipizzazione più opportune.
In questo studio, presentiamo un test in Real-Time PCR (qPCR TaqMan®) per la quantificazione del DNA specifico per applicazioni forensi, in grado di valutare simultaneamente sia la quantità di DNA nucleare che di DNA mitocondriale (mtDNA). Il saggio combina due target mtDNA e due target di DNA nucleare, con prodotti di amplificazione di dimensioni diverse (mtDNA=69bp e 143bp; DNA nucleare=71bp e 181bp), così da fornire informazioni sullo stato di degradazione del materiale genetico estratto. Inoltre, il test qPCR contiene un controllo positivo interno (IPC) per rilevare la presenza di potenziali inibitori. Tuttavia, a causa di un'interazione tra i primers del target mitocondriale da 69bp e la sonda del target nucleare da 71bp, riscontrata durante la validazione e l'ottimizzazione del saggio qPCR, è stato eliminato dal saggio il target da 71bp del DNA nucleare passando da una reazione pentaplex ad una reazione tetraplex. Il saggio è stato testato su diversi matrici biologiche, costituite da campioni forensi contenenti esigue quantità di DNA nucleare e/o DNA degradato, come ossa, denti, unghie, tessuti fissati in formalina e inclusi in paraffina (FFPE) e fusti dei capelli. I risultati di quantificazione ottenuti mediante il saggio tetraplex sono stati confrontati con i dati ottenuti sugli stessi campioni con altri sistemi di quantificazione esistenti in commercio e di uso comune nei laboratori di genetica forense.Quantification of human DNA plays a key role in forensic genetics. A more accurate estimate of the amount of human DNA is essential for planning and optimizing genotyping assays, as is an evaluation of the presence of PCR inhibitory substances present in forensic samples. Furthermore, for highly compromised samples, quantification can provide information about the DNA degradation status, directing the forensic analyst towards more appropriate genotyping strategies.
In this study, we present a Real-Time PCR assay (qPCR TaqMan®) for the quantification of DNA specific for forensic applications, able to assess simultaneously both the quantity of nuclear and mitochondrial DNA. The assay combines two mtDNA targets and two nuclear DNA targets, with amplification products of different sizes (mtDNA=69bp and 143bp; nuclear DNA=71bp and 181bp), in order to provide information on the degradation status of the extracted genetic material. In addition, the qPCR test contains an internal positive control (IPC) to detect the presence of potential inhibitors. However, due to an interaction between the 69bp mitochondrial target primers and the 71bp nuclear target probe, found during validation and optimization of the qPCR assay, the 71bp DNA target was removed from the assay passing from pentaplex to tetraplex reaction. The assay was tested on various biological matrices, consisting of forensic samples that contain small amounts of nuclear DNA and/or degraded DNA, such as bone, teeth, fingernails, formalin-fixed paraffin-embedded (FFPE) tissues and hair shafts. The quantification results obtained by the tetraplex assay have been compared with the data achieved on the same samples with other quantification systems commercially available and commonly used in forensic genetics laboratories
Evaluation of a microhaplotypes panel for forensic genetics using massive parallel sequencing technology
No full textMassive parallel DNA sequencing (MPS) makes it possible to explore a new type of genetic marker, known as microhaplotypes or microhaps. These loci were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Microhaplotypes loci, based on 89 loci in ALFRED, were selected and their genetic variations in 100 Italian individuals were evaluated by using MPS, in order to make inference about utility of a set of microhaps in forensic genetics. After MPS, the panel was reduced to 87 microhaps, comprised of 266 different SNPs and spread across 22 human autosomes. Genotype and haplotype frequencies were estimated, as well as the effective number of alleles at each locus (Ae), which relates to the usefulness of the locus in resolution of relationships and deconvolution of DNA mixtures. Overall, the Ae values for the 87 microhaps range from 1.010 to 8.344, with about 80% showing values greater than 2.0. Noteworthy, 32 microhaps display Ae values greater than 3.0 and 18 loci Ae above 4.0. To explore the suitability of microhaplotypes in mixture deconvolution, the probability of detecting a mixture, as a function of Ae, was inferred for different groups of loci. Considering the fourteen loci with Ae between 3.0 and 3.999 the probability of detecting a mixture was at least 0.99973, while considering the ten loci with Ae between 4.0 and 4.999 the probability was at least 0.99998. Moreover, when considering just the six loci with Ae between 5.0 and 5.999 the probability of detecting a mixture was at least 0.99984, while when considering just the two loci with Ae above 6 the probability was 0.97228. Combining these 32 MH loci, the theoretical probability of detecting a mixture was 0.999999999999973. These results make the subset of 32 loci with Ae above three informative for mixture resolution. The individual matching probabilities (PI) of the 87 microhaps ranged from 0.032 to 0.9802. Considering the 32 microhap loci with Ae greater than 3.0, the cumulative PI value was 1.6 × 10-33, while considering the 18 microhap loci with Ae above 4.0, the cumulative PI value was 2.34 × 10-21. Overall the results of this study confirmed the utility of microhaps in forensic genetics
Developmental validation of a multiplex qPCR assay for simultaneous quantification of nuclear and mitochondrial DNA
Full text linkQuantification of human DNA is key in forensic genetics. A more accurate estimate of the amount of DNA is essential for planning and optimising genotyping assays, as is evaluating the presence of PCR inhibitory substances and DNA degradation status. Multiplex qPCR assays are helpful in forensics because they can quantify different targets simultaneously, thus saving valuable samples, time, and labour. The aim of this study was to highlight the challenges in the developmental validation of a multiplex real-time PCR assay and the drawbacks encountered in translating a previously described and validated assay (SD quants) to a different technology by modifying the dye probes and reagent mix to be used in a different instrument. We developed a TaqMan probebased multiplex qPCR using reagents and fluorescent probes adapted for the Rotor-Gene 6000 instrument (QIAGEN, Hilden, Germany). The initial assay combined two mitochondrial DNA (mtDNA) and two nuclear DNA (nDNA) targets, with amplification products of different sizes (mtDNA = 69 and 143 bp; nDNA = 71 and 181 bp), to estimate the DNA degradation status and an internal positive control (IPC) to detect potential inhibitors. During the initial testing of the assay, we observed an interaction between the 69 bp mtDNA target and the 71 bp nDNA target probe, and experiments were conducted to resolve this issue without success. We removed the small nDNA target (71 bp) and changed from a 5-plex to a 4-plex qPCR assay (qMIND). The final tetraplex assay was tested on 105 forensic samples and/or small amounts of degraded DNA, such as bones, teeth, fingernails, formalin-fixed paraffin-embedded tissues (FFPE), and hair shaft samples. The quantification results were compared with data acquired from the same samples using another commercially available quantification system commonly used in forensic laboratories. In addition, the short tandem repeat (STR) profiles were investigated to determine their correlation with the quantitative values obtained. Overall, the qPCR assay was robust and reliable for DNA quantification in samples commonly used in forensic practice
Exploring the usefulness of microhaplotypes in forensic identification using massive parallel sequencing technology
No full textBACKGROUND: Microhaplotypes or microhaps (MH) were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, genotyping of degraded DNA, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. In order to make inference about a set of microhaps useful in forensic casework with low amount of degraded DNA and useful in kinship analysis, several microhaps were tested by massive parallel sequencing (MPS) assay.
METHODS: We have investigated the effectiveness of 29 microhaps in a set of real forensic samples together with artificially degraded DNAs. Moreover, we explore the informativeness of 87 microhaplotypes in relationship analysis through a simulation of different kinship testing scenarios typically encountered in forensic identification.
RESULTS: The MPS coverage analysis showed a good performance of the designed panel. Full profiles could be obtained with 0.1 ng of input DNA even with highly degraded samples. The increment of the number of PCR cycles does not result in an improvement in genotyping results in samples with low amounts of DNA, as the increase of drop-in and drop-out events were observed at 25 number of PCR cycles. No correlation between amplicons size and occurrence of drop-outs and drop-ins was observed. Kinship simulations showed that full siblings and half siblings relationships would be readily distinguished respect unrelated condition using the 87 microhaps panel.
CONCLUSIONS: Results shown that microhaps could be a powerful tool for individual identification, relationship resolution and that they are sensitive and reliable in degraded DNA typing
Validation of a universal DNA extraction method for human and microbiAL DNA analysis
No full textThe study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). Before a new technology is accepted by the forensic science, it requires an initial validation phase. The aim of our study was to evaluate if the DNA IQTM Casework Pro Kit for Maxwell® 16 (Promega) is suitable for microbial DNA extraction, without modifications. Ten bacterial strains were selected and subjected to the GenElute Bacterial Genomic DNA extraction protocol (Sigma-Aldrich) and to the DNA IQTM Casework Pro Kit for Maxwell® 16 protocol. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Reporter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the Promega kit, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited
Performance of a massive parallel sequencing microhaplotypes assay on degraded DNA
Full text linkMassively parallel sequencing (MPS) has allowed to analyze a new type of forensic genetic marker, known as microhaplotypes (MHs). MHs appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Moreover, MHs are potentially suitable for the analysis of degraded DNA samples. We designed a new panel of 29 MHs for MPS assay, with amplicons sizes below 180 bp and we investigated its effectiveness with low amounts of degraded samples. We genotyped a set of real forensic samples together with a set of artificially degraded DNAs. Also, a sensitivity test was assessed by a set of 2800 M DNA dilutions. The Depth of Coverage (DoC) were uniform across all 29 loci, in spite of amplicons size. Genotyping results shown that full profiles can be obtained even in highly degraded samples when the amount of template range from 0.1 to 5.0 ng. Finally, the increase of the number of PCR cycles did not provide an improvement in typing results of low amounts of degraded samples as, in front of higher number of typed loci, higher frequencies of artefacts leading to mistyping are found at 25 cycles
Microhaplotypes in forensic genetics: From exploration to application in degraded DNA specimens
Full text linkMicrohaplotypes have emerged as powerful forensic markers over the past decade. This paper sets out the development of a MPS panel of microhaps and its potential for application to identification, analysis of degraded DNA, ancestry inference, and identification of close biological relationships. To make it more effective when dealing with fragmented DNA, the MPS assay was designed to ensure a reduced amplicon size of less than 140 bp. After MPS assay validation, a panel of 76 microhaps, comprised of 299 different SNPs and spread across the autosomal human genome, was established. A total of 102 Italian individuals were analyzed to estimate the genotype and haplotype frequencies. The effective number of alleles at each locus (Ae) for the Italian population ranges from 1.926 to 6.187, with 59 MHs that have values greater than 3.0. The matching probability (PI) ranges from 0.055 to 0.345 and the cumulative PI value is 11.763E-66. Complete and reliable profiles were obtained with as little as 0.05 ng. The MHs panel was then validated on real forensic specimens chosen on the basis of their DNA content and degradation level. The majority of the casework samples analyzed showed complete or nearly complete MH profiles even in degraded samples. To assess the informative power of MH profiles in forensic casework, probabilistic genotyping on partial MH profiles has been used. The resulting likelihood ratio values range from 7.84E+09 to 2.70E+34, thus defining an extremely strong support for the hypothesis that the genetic profile in a casework sample comes from the reference sample. Pairwise kinship simulations using allele frequencies from Italian population samples showed that full- and half-sibling relationships can be readily distinguished from unrelated individuals. For evaluation of the 76 MH panel's utility for ancestry informativeness, PCA and STRUCTURE analyses are also presented comparing the newly collected sample from Ancona Italy with the 26 populations of the 1000 Genomes Project. The results of the analysis confirmed the effectiveness of these short microhaplotypes in typing, with high sensitivity, samples with highly degraded DNA typically encountered in forensic cases
Sudden Cardiac Death and Channelopathies: What Lies behind the Clinical Significance of Rare Splice-Site Alterations in the Genes Involved?
Full text linkBackground and objectives: Sudden cardiac death (SCD) is a natural and unexpected death of cardiac origin that occurs within 1 h from the onset of acute symptoms. The major leading causes of SCD are cardiomyopathies and channelopathies. In this review, we focus on channelopathies, inherited diseases caused by mutations affecting genes encoding membrane ion channels (sodium, potassium or calcium channels) or cellular structures that affect Ca2+ availability. The diagnosis of diseases such as long QT, Brugada syndrome, short QT and catecholaminergic polymorphic ventricular tachycardia (CPVT) is still challenging. Currently, genetic testing and next-generation sequencing allow us to identify many rare alterations. However, some non-coding variants, e.g., splice-site variants, are usually difficult to interpret and to classify. Methods: In our review, we searched for splice-site variants of genes involved in channelopathies, focusing on variants of unknown significance (VUSs) registered on ClinVar up to now. Results: The research led to a high number of splice-site VUSs of genes involved in channelopathies, suggesting the performance of deeper studies. Conclusions: In order to interpret the correlation between variants and pathologies, we discuss experimental studies, such as RNA sequencing and functional analysis of proteins. Unfortunately, as these in vitro analyses cannot always be performed, we draw attention to in silico studies as future perspectives in genetics. This review has the aim of discussing the potential methods of detection and interpretation of VUSs, bringing out the need for a future reclassification of variants with currently unknown significance
MicroRNAs as diagnostic biomarkers in cardiovascular diseases that cause sudden cardiac death: comparison between methods used in discovery and validation phases in major research studies
No full textINTRODUCTION: MicroRNAs (miRNAs) are a non-coding RNA class of about 22 nucleotides that regulate gene expression by complementarily binding the 3’-untraslated regions of mRNA; thus, causing the inhibition of translation or mRNA degradation. A different expression profile of miRNAs (upregulated or downregulated) could lead to an imbalance in gene expression. EVIDENCE ACQUISITION: In recent years, many studies have focused on miRNAs and their role as biomarkers in diseases with difficult and inconclusive diagnoses. One of the most challenging diagnoses in the forensic field is sudden cardiac death (SCD) diagnosis which still has no comprehensive answers to the causes that determine the death of an apparently healthy subject. EVIDENCE SYNTHESIS: Thus, it could be interesting to study new biomarkers in genetic tests such as miRNAs. We have collected the most interesting studies in literature, focusing on the quantification methods used for the detection and validation of miRNAs with a potential role as biomarkers in arrhythmogenic cardiomyopathy, one of the most frequent pathological causes of SCD. CONCLUSIONS: In this review we will discuss the scientific logical path that starts from a discovery phase, using screening methods to select a group of differentially expressed miRNAs, up to a validation phase, which selects only informative miRNAs with a potential role as biomarkers
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