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Forensic Science, Medicine, and... Mar 2023Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess...
Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.
Topics: Humans; DNA; DNA Damage; DNA Degradation, Necrotic; DNA Fingerprinting; Microsatellite Repeats; Real-Time Polymerase Chain Reaction; Blood Specimen Collection
PubMed: 36355320
DOI: 10.1007/s12024-022-00549-3 -
Methods in Molecular Biology (Clifton,... 2023In the field of forensic science, the DNA extraction of bone is utilized in investigations involving mass disasters, unidentified remains, and missing persons. However,...
In the field of forensic science, the DNA extraction of bone is utilized in investigations involving mass disasters, unidentified remains, and missing persons. However, bone samples can be challenging samples due to their exposure to extreme environmental conditions over long periods of time. The use of an effective DNA extraction method to properly isolate and purify the DNA is essential for bone samples. This chapter describes the DNA extraction of bone samples through a total demineralization protocol, which aims to entirely dissolve the bone matrix in order to access the DNA molecules.
Topics: DNA Fingerprinting; Microsatellite Repeats; Bone and Bones; DNA; Bone Matrix
PubMed: 37439977
DOI: 10.1007/978-1-0716-3295-6_6 -
Methods in Molecular Biology (Clifton,... 2023The GlobalFiler™ PCR Amplification Kit is one of the most sensitive kits that exist today that makes the PCR amplification of human DNA possible. PCR amplification...
The GlobalFiler™ PCR Amplification Kit is one of the most sensitive kits that exist today that makes the PCR amplification of human DNA possible. PCR amplification using this specific kit makes millions of copies of 24 specific target sequences in the DNA, called markers or loci. This kit is a 6-dye, short tandem repeat (STR) multiplex assay kit that has a synthetic mix of primers and single-stranded oligonucleotides that are combined with DNA samples and then subjected to 29 or 30 cycles of denaturing, annealing, and extension, as per laboratory protocol. Methods for instrument operation will vary depending on the thermal cycler instrument model that is used. Nevertheless, the GlobalFiler™ PCR Amplification Kit has proven to be a very useful tool to DNA analysts, amplifying extremely low quantities of DNA, making it possible to detect partial, if not full, genetic profiles from a wide range of sample types. This chapter discusses the typical preparation and PCR amplification of human forensic DNA samples, using the GlobalFiler™ PCR Amplification Kit.
Topics: Humans; DNA Fingerprinting; Polymerase Chain Reaction; Microsatellite Repeats; DNA; DNA Primers
PubMed: 37439986
DOI: 10.1007/978-1-0716-3295-6_15 -
International Journal of Legal Medicine Sep 2022The choice of skeletal element types and their intra-bone parts is important because of differences in DNA preservation, and this must be considered when sampling bones...
The choice of skeletal element types and their intra-bone parts is important because of differences in DNA preservation, and this must be considered when sampling bones for DNA testing. When incomplete skeletons are found, ribs and vertebrae have been shown to be the most suitable for genetic identification of bones from the torso. This study compares the preservation of DNA between 12th thoracic vertebrae and first ribs to determine which bone type is more suitable for genetic typing. The study analyzed 35 12th thoracic vertebrae and 29 first ribs from one mass grave from the Second World War with commingled skeletal remains excavated. Bone DNA preservation was estimated by measuring nuclear DNA concentration and its degradation and through short tandem repeat (STR) typing success. Previous studies performed on aged skeletal remains have shown that the DNA content of the first ribs and 12th thoracic vertebrae has high intra-bone variability, and this was considered when sampling the bones. After full demineralization extraction, the PowerQuant System (Promega) was used to measure the quantity and quality of DNA, and the GlobalFiler kit (Applied Biosystems) was used for STR typing. The results showed that DNA yield and degradation and STR typing success exhibited no statistically significant difference between first ribs and 12th thoracic vertebrae, and there was no intra-individual difference when comparing only paired bones from the same individuals. Consequently, with intra-bone DNA variability considered, the first ribs or the 12th thoracic vertebrae can be selected when sampling to genetically identify the skeletal remains of highly degraded torsos. HIGHLIGHTS: The first ribs and thoracic vertebrae are the most suitable bones for sampling from the torso. The proximal part of first rib and posterior vertebral column of the 12th thoracic vertebrae yielded the most DNA. The first ribs were compared with the 12th thoracic vertebrae, and the sampling process considered intra-bone DNA variability. The quality and quantity of nuclear DNA and success of STR typing were measured. The first ribs yielded the same DNA yields as well as STR typing success as the 12th thoracic vertebrae. When only the torso is present, it is not of high importance whether the first ribs or the 12th thoracic vertebrae are collected.
Topics: Aged; Body Remains; DNA; DNA Fingerprinting; Humans; Microsatellite Repeats; Ribs; Spine; Thoracic Vertebrae
PubMed: 35729437
DOI: 10.1007/s00414-022-02860-8 -
Genes Jan 2022The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service...
The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.
Topics: Body Remains; DNA Fingerprinting; DNA, Ancient; Forensic Genetics; High-Throughput Nucleotide Sequencing; Humans; Korean War; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; World War II
PubMed: 35052469
DOI: 10.3390/genes13010129 -
Journal of Forensic Sciences May 2023Forensic "touch" DNA samples are low-quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can...
Forensic "touch" DNA samples are low-quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can include DNA from primary contributors who directly touched the surface, as well as secondary contributors whose DNA was transferred to the surface through an intermediary. It is difficult to determine the type of transfer, or how often and under what conditions DNA transfer occurs. In this paper, we present an innovative protocol that combines (1) a paired male and female transfer DNA experimental design in which the presence of male DNA indicates secondary transfer and (2) a cost-effective quantitative PCR (qPCR) assay of a sex-specific region in the Amelogenin gene to detect male and female DNA. We evaluate the ability of the Amelogenin qPCR assay to detect low concentrations of male and female DNA in mixed samples. We also test experimental DNA samples using our transfer DNA protocol to differentiate primary and secondary DNA transfer. Male DNA was detected in the majority of known mixed samples, even in samples with 4× more female DNA-this result demonstrates the ability to detect low concentrations of male DNA and the presence of secondary transfer DNA in our experimental design. Primary DNA transfer was detected in 100% of our experimental trials and secondary DNA transfer was detected in 37.5% of trials. Our innovative protocol mimics realistic case scenarios to establish rates of primary and secondary DNA transfer in an inexpensive and simplified manner.
Topics: Humans; Male; Female; Pilot Projects; Research Design; Amelogenin; Polymerase Chain Reaction; DNA; DNA Fingerprinting
PubMed: 36975017
DOI: 10.1111/1556-4029.15243 -
Science & Justice : Journal of the... Mar 2022Successful DNA typing of forensically relevant evidence is reliant on both the quality and quantity of biological material recovered from a crime scene. In geographical...
Successful DNA typing of forensically relevant evidence is reliant on both the quality and quantity of biological material recovered from a crime scene. In geographical areas of the world exposed to cold climates, it is not uncommon for biological evidence to encounter a diversity of challenging surfaces and environments, including snowy surfaces. Currently, there is no standard protocol for recovery of bloodstain evidence in snow and very few publications exploring adequate methods of recovering biological evidence from snowy surfaces. In this study, three common substrates (e.g., cotton swabs, FTA paper, and untreated filter paper) utilized by investigators for evidence recovery were evaluated for their ability to recover human blood (DNA) evidence from snow that would be viable for traditional forensic DNA typing. Each biological sample was extracted and quantified to evaluate the quality and quantity of DNA recovered. All samples yielded sufficient non-degraded DNA to proceed with DNA profiling, where complete DNA profiles were generated from each collection substrate. The experimental findings presented herein demonstrate that the ability to recover viable DNA from human blood collected on surface snow is possible using all three collection methods tested.
Topics: DNA; DNA Fingerprinting; Humans; Microsatellite Repeats; Snow; Specimen Handling
PubMed: 35277228
DOI: 10.1016/j.scijus.2022.01.001 -
Genes Mar 2024The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The... (Review)
Review
The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.
Topics: Humans; Polymerase Chain Reaction; Forensic Sciences; DNA Fingerprinting; DNA; Forensic Genetics
PubMed: 38674373
DOI: 10.3390/genes15040438 -
Science & Justice : Journal of the... Jul 2021"Shedder status" or "shedder type" are commonly used terms that categorise an individual based on their ability to deposit "touch" DNA via direct contact with a surface....
"Shedder status" or "shedder type" are commonly used terms that categorise an individual based on their ability to deposit "touch" DNA via direct contact with a surface. However, it is not yet clear how best to categorise an individual into a shedder class, or how to allocate a shedder score on a sliding scale. This study considers categorisation of participants into discrete shedder categories based on DNA quantity and profile quality data, the maintenance of their shedder status over an extended period, and explores whether different methods of deposition or collection directly from hands or other body areas are interchangeable and/or more appropriate means of determining an individual's shedder status. The shedder categorisation of participants was possible from their handprints and remained unchanged over three years. Washing hands had limited impact and shedder categorisation was not readily possible from samples collected directly from hands, other body areas or gloves after wearing gloves for a set duration. Use of consecutive deposits may assist in establishing a participant's shedder status. As shedder categorisation may be of relevance during activity level assessments further efforts towards the ability to do so are necessary.
Topics: DNA; DNA Fingerprinting; Hand; Humans; Touch
PubMed: 34172128
DOI: 10.1016/j.scijus.2021.03.004 -
International Journal of Molecular... Jun 2022Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic... (Review)
Review
Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic DNA amounts as small as 125 pg. Yet these techniques have reached their limits when it comes to the analysis of traces such as fingerprints or single cells. One suggestion to overcome these limits has been the usage of whole genome amplification (WGA) methods. These methods aim at increasing the copy number of genomic DNA and by this means generate more template DNA for subsequent analyses. Their application in forensic contexts has so far remained mostly an academic exercise, and results have not shown significant improvements and even have raised additional analytical problems. Until very recently, based on these disappointments, the forensic application of WGA seems to have largely been abandoned. In the meantime, however, novel improved methods are pointing towards a perspective for WGA in specific forensic applications. This review article tries to summarize current knowledge about WGA in forensics and suggests the forensic analysis of single-donor bioparticles and of single cells as promising applications.
Topics: DNA; DNA Fingerprinting; Genome, Human; Humans; Microsatellite Repeats; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction
PubMed: 35806097
DOI: 10.3390/ijms23137090